Biomedical subjects

Lutz Jermutus

Publications and source records attributed to Lutz Jermutus.

12 recordsLinked to original sources

PubMed2006

Harnessing phage and ribosome display for antibody optimisation.

Therapeutic antibodies have become a major driving force for the biopharmaceutical industry; therefore, the discovery and development of safe and efficacious antibody leads have become competitive processes. Phage and ribosome display are ideal tools for the generation of such molecules and have already delivered an approved drug as well as a multitude of clinical candidates. Because they are capable of searching billions of antibody variants in tailored combinatorial libraries, they are particularly applicable to potency optimisation. In conjunction with targeted, random or semi-rational mutagenesis strategies, they deliver large panels of potent antibody leads. This review introduces the two technologies, compares them with respect to their use in antibody optimisation and highlights how they can be exploited for the successful and efficient generation of putative drug candidates.

Antibodies↗

PubMed2006

Parallel, high-throughput purification of recombinant antibodies for in vivo cell assays.

We describe a method for high-throughput, parallel purification of secreted proteins to analyse large numbers of protein samples in cell-based assays for the discovery of protein therapeutics. The procedure is generic and capable of 96 parallel purifications and compatible, in both yield and purity, with a wide assay range. By optimising expression and purification steps as well as using novel hardware, in particular a chromatography press capable to purify target proteins from viscous media, we exemplify the process for the generation of single-chain Fv antibody fragments (scFv) and the purification of full-length IgG. The described process can operate robustly with a throughput of over 2,000 samples per month.

Antibodies, Monoclonal↗

PubMed2006

Probing a protein-protein interaction by in vitro evolution.

In this study, we used in vitro protein evolution with ribosome and phage display to optimize the affinity of a human IL-13-neutralizing antibody, a therapeutic candidate for the treatment of asthma, >150-fold to 81 pM by using affinity-driven stringency selections. Simultaneously, the antibody potency to inhibit IL-13-dependent proliferation in a cell-based functional assay increased 345-fold to an IC50 of 229 pM. The panoply of different optimized sequences resulting from complementarity-determining region-targeted mutagenesis and error-prone PCR using ribosome display was contrasted with that of complementarity-determining region-targeted mutagenesis alone using phage display. The data highlight the advantage of the ribosome-display approach in identifying beneficial mutations across the entire sequence space. A comparison of mutation hotspots from in vitro protein evolution to knockout mutations from alanine scanning demonstrated that in vitro evolution selects the most appropriate positions for improvements in potency without mutating any of the key residues within the functional paratope.

Amino Acid Sequence↗

PubMed2006

Exploiting directed evolution for the discovery of biologicals.

Protein-based drugs offer unique advantages over small-molecule drugs in terms of both discovery and therapeutic use. The advent of recombinant DNA technology enabled the production of recombinant proteins and the generation of partially or fully human monoclonal antibodies, and continued developments in molecular biology have provided powerful approaches to generate improved proteins with more drug-like features. In this review, the benefits of using an evolutionary approach to biologicals drug discovery are discussed, with emphasis on the use of in vitro evolution technologies, such as ribosome display.

Animals↗

PubMed2005

Affinity maturation of a V(H)H by mutational hotspot randomization.

V(H)Hs from naive libraries have dissociation constants (K(D)s) in the low micromolar range and thus, for most antibody applications, their intrinsic affinities need to be improved significantly. Non-targeted in vitro affinity maturation approaches based on indiscriminate randomization of complementarity-determining region (CDR) residues or random mutagenesis of conventional antibody variable domains have been shown to improve the affinity of recombinant antibodies by 450- to over 6000-fold. A different, targeted approach based on selective randomization of CDR codons containing AGY/RGYW nucleotide mutational hotspots i.e., "hotspot codons", also promises to be very efficient for improving antibody affinities. Here we employed the latter approach for improving the affinity of PTH22, a parathyroid hormone (PTH)-derived peptide-specific V(H)H that was isolated from a naive llama phage display library. A PTH22 mutant ribosome display library was constructed by randomizing nine CDR2 and CDR3 hotspot codons. The affinity improvement of the lead binder was 30-fold, which seems somewhat low in view of the large number of randomized hotspot codons. Nucleotide sequence analyses of PTH22 and 23 naive V(H)Hs suggested that many AGY/RGYW mutational hotspots are not affinity mutational hotspots but play a role in V(H)H solubility, structure, and deletion/insertion events. Our results indicate that the mutagenesis approach described here is beneficial in terms of yielding moderate increases in affinity while fine-tuning physical properties of an antibody.

Amino Acid Motifs↗

PubMed2004

Combinatorial protein biochemistry for therapeutics and proteomics.

This paper reviews progress towards the customised generation of proteins for applications as therapeutics, diagnostics and in proteomics. It particularly focuses on combinatorial approaches where proteins with pre-defined biochemical and biophysical characteristics are selected from large variant libraries.

Animals↗

PubMed2003

Selection of hapten-specific single-domain antibodies from a non-immunized llama ribosome display library.

Picloram-specific variable fragments (V(HH)s) of heavy chain antibodies (HCAbs) were selected from a nai;ve-llama library using ribosome display technology. A cDNA library of V(HH)s was constructed from lymphocytes of a non-immunized llama and engineered to allow in vitro transcription and translation. With no stop codons present on the transcripts, trimeric complexes of ribosomes, mRNAs and nascent peptides were produced for affinity selection, i.e. panning. After three cycles of panning, seven different V(HH)s all belonging to the V(HH) subfamily 1 were isolated. Following another three cycles of selection, only two of the seven V(HH)s persisted. A comparison of these two sequences with known sequences in the literature suggests that point mutations may have been introduced into the DNA pool during PCR amplification steps of library construction, panning and/or cloning. Three separate point mutations causing three independent amino acid changes (nonsynonomous mutations) accumulated in the same sequence and enriched throughout the selection protocol, suggesting that these changes confer binding advantages. Surface plasmon resonance (SPR) analysis was used to determine binding kinetics of the two clones (3-1D2 and 3-1F6) representing the two different sets of isolated complementarity determining region (CDR)3s. Measured K(D)s were 3 and 254 muM, respectively. The results indicate that ribosome display technology can be used to efficiently isolate hapten-specific antibody (Ab) fragments from a nai;ve library and concurrently introduce diversity to the selected pool thereby facilitating molecular evolution. Ribosome display technology can compensate for the limited diversity of a V(HH) nai;ve library and provide an unlimited source of affinity-matured immunoactive reagents in vitro.

Amino Acid Sequence↗

PubMed2003

Current methods for the generation of human antibodies for the treatment of autoimmune diseases.

Autoimmune diseases are a significant area of unmet medical need in the Western World, but human antibodies are an emerging drug class that could address this demand. Some autoimmune diseases, such as rheumatoid arthritis, are currently benefiting from antibody treatment and new and existing technologies for antibody generation could facilitate the production of effective human antibodies as future drug candidates for other autoimmune diseases. Several methods of generating human antibodies for use as therapeutics have been established, the most commonly used being phage display and transgenic mouse technologies and more recently, cell-free display technologies have also emerged. In this review, we explain the principles behind the various methods of antibody generation and highlight some potential benefits of certain approaches in the context of treatment of autoimmune disease.

Animals↗

PubMed2002

In vitro selection for catalytic activity with ribosome display.

We report what is, to our knowledge, the first in vitro selection for catalytic activity based on catalytic turnover by using ribosome display, a method which does not involve living cells at any step. RTEM-beta-lactamase was functionally displayed on ribosomes as a complex with its encoding mRNA. We designed and synthesized a mechanism-based inhibitor of beta-lactamase, biotinylated ampicillin sulfone, appropriate for selection of catalytic activity of the ribosome-displayed beta-lactamase. This derivative of ampicillin inactivated beta-lactamase in a specific and irreversible manner. Under appropriate selection conditions, active RTEM-beta-lactamase was enriched relative to an inactive point mutant over 100-fold per ribosome display selection cycle. Selection for binding, carried out with beta-lactamase inhibitory protein (BLIP), gave results similar to selection with the suicide inhibitor, indicating that ribosome display is similarly efficient in catalytic activity and affinity selections. In the future, the capacity to select directly for enzymatic activity using an entirely in vitro process may allow for a significant increase in the explorable sequence space relative to existing strategies.

Ampicillin↗

PubMed2002

Ligand binding of a ribosome-displayed protein detected in solution at the single molecule level by fluorescence correlation spectroscopy.

Interaction of a single-chain antibody fragment (scFv) with its cognate antigen while still attached to the ribosome was studied by fluorescence correlation spectroscopy (FCS). In experiments with purified scFv, FCS was capable of resolving the difference in diffusion time between free and antibody-bound labelled antigen. Ribosome-displayed antibody fragments generated by in vitro translation, in which neither the protein nor the mRNA leaves the ribosome owing to the absence of a stop codon and stabilizing buffer conditions, could be shown to specifically bind the antigen. The antibody-antigen interaction was specific, as shown by inhibition or displacement with unlabelled antigen and by control experiments with a non-cognate antibody fragment.

Amino Acid Sequence↗

PubMed2002

Concepts in antibody phage display.

This paper introduces the reader to antibody phage display and its use in combinatorial biochemistry. The focus is on overviewing phage display formats, library design and selection technology, which are the prerequisites for the successful isolation of specific antibody fragments against a diverse set of target antigens.

Antibodies↗

PubMed2002

Phage Display Technologies - SMi Conference. 23-24 January 2002, London, UK.

This fairly small meeting, with about 50 attendees, covered all areas of antibody engineering from basic technologies to applications in both therapeutic and diagnostic areas. Despite the fact that nearly all speakers came from commercial organizations, the data presented were solid and highly informative. While new technologies, such as yeast or ribosome display, are integrating into the drug discovery process, vaccine technology looks like a promising alternative to human antibody therapy. New advances in the field, especially in technology, are primarily governed by the tight intellectual property situation.

Journal Article↗