Designing Human Antibodies by Phage Display

Abstract

ever, are recognized as foreign proteins by humans and therefore induce immune reactions against the therapeutic reagent (human anti-mouse antibody; HAMA) [3]. A solution to this problem was only brought by recombinant DNA technology. Using genetic engineering, the murine sequences were exchanged in parts of the molecule by the homologous human sequences. The result were 'chimeric' (with murine constant domains replaced by human constant domains) or 'humanized' antibodies (only the antigen-binding loops / complementarity determining regions (CDRs) were transferred to human variable region frameworks) [4,5]. These engineered mouse antibodies dominated the first decade of therapeutic antibody approvals, while today it is preferred to use completely human antibodies from the start, thanks to a number of novel technologies allowing to identify them without the necessity to immunize humans. These are on the one hand transgenic animals, typically mice or rats, which carry genetically introduced human immunoglobulin loci while their own antibody genes were knocked out [6][7][8]. Consequently, after immunization they utilize this sequence repertoire to generate IgG from human germ-line sequences, thereby allowing to generate monoclonals by means of classical hybridoma technology. An even more radical way was introduced by the development of antibody phage display, which allowed for the first time to generate human antibodies completely in the test tube and without immunization. Inspired by the work of G.P. Smith on peptide display using filamentous phage [9], antibody phage display was development independently in Heidelberg by Breitling and Dübel [10], in Cambridge by McCafferty et al. [11], and in California by Barbas et al. [12]. The method is based on a physical link between function (antigen binding) and information (antibody gene) in a nanoparticle (the phage virus particle). To achieve this, the antigen binding parts of the antibodies, either as Fab (fragment antigen binding) [13,14] or scFv (single chain fragment variable) [15][16][17], are genetically linked to the surface protein III (pIII) of the M13 phage and thus expressed on the surface of the virus particle. Mixtures of such phage particles, each enKeywords Human monoclonal antibodies · Phage display · Immunotherapy · In vitro evolution · Panning · scFv Summary With six approved products and more than 60 candidates in clinical testing, human monoclonal antibody discovery by phage display is well established as a robust and reliable source for the generation of therapeutic antibodies. While a vast diversity of library generation philosophies and selection strategies have been conceived, the power of molecular design offered by controlling the in vitro selection step is still to be recognized by a broader audience outside of the antibody engineering community. Here, we summarize some opportunities and achievements, e.g., the generation of antibodies which could not be generated otherwise, and the design of antibody properties by different panning strategie...

Human antibodies neutralizing diphtheria toxin in vitro and in vivo

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Bivalent binding of a fully human IgG to the SARS-CoV-2 spike proteins reveals mechanisms of potent neutralization

“…In vitro antibody selection against pathogens from naïve combinatorial libraries can yield various classes of antigen-specific binders that are distinct from those evolved from natural infection [1][2][3][4] . Also, rapid neutralizing antibody discovery can be made possible by a strategy that selects for those interfering with pathogen and host interaction 5 The ongoing COVID-19 pandemic, caused by a novel coronavirus SARS-CoV-2 originating in Wuhan, China, at the end of 2019, is one of the world's most devastating pandemics in living memory.…”

“…In addition to those derived from natural infection or immunization, potent neutralizing antibodies against pathogens can be isolated by in vitro selection from highly diverse combinatorial human libraries [1][2][3] . Interestingly, some of such antibodies often came from different antibody subtypes and recognized different epitopes 31 .…”

Applying Antibodies Inside Cells: Principles and Recent Advances in Neurobiology, Virology and Oncology

“…Methods to generate binders are briefly summarized in Table 2. For more detailed information, interested readers are referred to reviews on technologies for generating binders, such as display technologies including phage display, yeast display, ribosome display and bacterial display [24][25][26][27][28][29]. Among the group of recombinant binders, there are differences in species and in biochemical properties, which might be relevant for their use as intrabodies or other purposes.…”