Affinity Maturation of Antibody Fragments by Phage Display for the Development of therapeutic IgGs

Monoklonale Antikörper werden für die Therapie unterschiedlicher Krankheiten eingesetzt, darunter sind vor allem schwerwiegende Erkrankungen wie Krebs oder Autoimmunkrankheiten. Für diesen Zweck werden bisher vorwiegend IgGs verwendet. Die in vitro Affinitätsreifung findet dagegen hauptsächlich in den weniger komplexen Fragmenten der vollständiger Antikörper statt, da deren Expression durch prokaryotische Produktionswirte möglich ist. In der vorliegenden Arbeit wurde gezeigt, dass das Antikörperformat, das zur Phagen-Display basierten Affinitätsreifung eingesetzt wird, die Bindungsstärke der resultierenden IgGs beeinflusst. Die Konvertierung affinitätsgereifter Antikörper in das IgG-Format kann mit einem Affinitätsverlust einhergehen, so dass die konvertierten Antikörper keine erhöhte Affinität gegenüber dem ursprünglichen IgG haben. Diese Beobachtung wurde für den Antikörper SH313-F9 in den Formaten scFv und scFabdC gemacht, bei denen wahrscheinlich Mutationen bevorzugt werden, die nur durch die Präsenz des Petidlinkers zu erhöhten Affinitäten gegenüber dem Ausgangsantikörper führten. Im Gegensatz dazu behielten die im FabdC-Format gereiften Antikörper ihre verstärkte Antigenbindung auch im IgG-Format bei. Durch die Deletion der jeweils C-terminalen Cysteine des Fabs wurde die Präsentation funktioneller Antikörperfragmente auf der Oberfläche von Phagen im Vergleich zum Fab-Format verbessert. Darüber hinaus wurden in dem resultierenden FabdC-Fragment im Gegensatz zum Fab affinitätsgereifte Varianten selektiert. Aufbauend auf den affinitätgesteigerten Antikörpern wurden zwei Shuffling-Methoden angewandt, um die Affinitäten der IgGs weiter zu erhöhen. Während das Chain Shuffling nicht zu einer weiteren Steigerung der Affinitäten der IgGs führte, wurde dies und teilweise auch eine gesteigerte Temperaturstabilität durch das DNA-Shuffling mit anschließender Phagen-Display Selektion im FabdC-Format erreicht. Die dadurch erfolgreich generierten, affinitätsgereiften IgGs waren eine Bestätigung der Konvertierung affinitätsgereifter FabdC in IgGs ohne den Verlust von Affinität.Monoclonal antibodies have become an important therapeutic tool for the treatment of various diseases, mostly cancer and autoimmune diseases. For this purpose nearly all antibodies are provided as IgGs. In vitro affinity maturation is mostly carried out in less complex fragments of full length antibodies, because they can be expressed by prokaryotic production hosts. In this study it was demonstrated that the antibody format used for phage display based affinity maturation influences the affinity of the corresponding IgG. The conversion of affinity matured antibody fragments into IgG can be accompanied with loss of affinity resulting in IgGs with decreased antigen binding compared to the parental clone. This behavior was shown for SH313-F9 scFv and scFabdC, possibly due to mutations, that led to enhanced affinities compared to the original antibody only in the presence of the peptide linker. In contrast, affinity matured FabdC retained their enhanced affinity as IgG. The deletion of c-terminal cysteines led to enhanced functional expression on the phage surface and allowed the generation of affinity matured antibodies compared to Fab. Based on the affinity matured antibodies, two shuffling methods were employed to further enhance the affinty of the IgGs. Chain shuffling of affinity matured antibody fragments converted to IgG did not lead to further enhancement of affinity. In contrast, IgGs generated by DNA shuffling had enhanced affinities and partially enhanced temperature stability compared to former affinity matured IgGs. The generation of affinity matured IgGs by DNA shuffling followed by FabdC based phage display selection was a confirmation for the conversion of affinity matured FabdC into IgG without loosing affinity

Efficient production of soluble recombinant single chain Fv fragments by a Pseudomonas putida strain KT2440 cell factory

<p>Abstract</p> <p>Background</p> <p>Recombinant antibody fragments have a wide range of applications in research, diagnostics and therapy. For many of these, small fragments like single chain fragment variables (scFv) function well and can be produced inexpensively in bacterial expression systems. Although <it>Escherichia coli </it>K-12 production systems are convenient, yields of different fragments, even those produced from codon-optimized expression systems, vary significantly. Where yields are inadequate, alternative production systems are needed. <it>Pseudomonas putida </it>strain KT2440 is a versatile biosafety strain known for good expression of heterologous genes, so we have explored its utility as a cell factory for production of scFvs.</p> <p>Results</p> <p>We have generated new broad host range scFv expression constructs and assessed their production in the <it>Pseudomonas putida </it>KT2440 host. Two scFvs bind either to human C-reactive protein or to mucin1, proteins of significant medical diagnostic and therapeutic interest, whereas a third is a model anti-lysozyme scFv. The KT2440 antibody expression systems produce scFvs targeted to the periplasmic space that were processed precisely and were easily recovered and purified by single-step or tandem affinity chromatography. The influence of promoter system, codon optimization for <it>P. putida</it>, and medium on scFv yield was examined. Yields of up to 3.5 mg/l of pure, soluble, active scFv fragments were obtained from shake flask cultures of constructs based on the original codon usage and expressed from the <it>Ptac </it>expression system, yields that were 2.5-4 times higher than those from equivalent cultures of an <it>E. coli </it>K-12 expression host.</p> <p>Conclusions</p> <p><it>Pseudomonas putida </it>KT2440 is a good cell factory for the production of scFvs, and the broad host range constructs we have produced allow yield assessment in a number of different expression hosts when yields in one initially selected are insufficient. High cell density cultivation and further optimization and refinement of the KT2440 cell factory will achieve additional increases in the yields of scFvs.</p

Structural differences of amyloid-beta fibrils revealed by antibodies from phage display

Background: Beside neurofibrillary tangles, amyloid plaques are the major histological hallmarks of Alzheimer's disease (AD) being composed of aggregated fibrils of beta-amyloid (A beta). During the underlying fibrillogenic pathway, starting from a surplus of soluble A beta and leading to mature fibrils, multiple conformations of this peptide appear, including oligomers of various shapes and sizes. To further investigate the fibrillization of beta-amyloid and to have tools at hand to monitor the distribution of aggregates in the brain or even act as disease modulators, it is essential to develop highly sensitive antibodies that can discriminate between diverse aggregates of A beta. Results: Here we report the generation and characterization of a variety of amyloid-beta specific human and human-like antibodies. Distinct fractions of monomers and oligomers of various sizes were separated by size exclusion chromatography (SEC) from A beta 42 peptides. These antigens were used for the generation of two A beta 42 specific immune scFv phage display libraries from macaque (Macaca fascicularis). Screening of these libraries as well as two naive human phage display libraries resulted in multiple unique binders specific for amyloid-beta. Three of the obtained antibodies target the N-terminal part of A beta 42 although with varying epitopes, while another scFv binds to the a-helical central region of the peptide. The affinities of the antibodies to various A beta 42 aggregates as well as their ability to interfere with fibril formation and disaggregation of preformed fibrils were determined. Most significantly, one of the scFv is fibril-specific and can discriminate between two different fibril forms resulting from variations in the acidity of the milieu during fibrillogenesis. Conclusion: We demonstrated that the approach of animal immunization and subsequent phage display based antibody selection is applicable to generate highly specific anti beta-amyloid scFvs that are capable of accurately discriminating between minute conformational differences