COR-101, ein menschlicher Antikörper gegen COVID-19.

COR-101 is a fully human, Fc silenced IgG that was discovered by antibody phage display. It reduced the SARS-CoV-2 virus load in the lung by more than 99 percent in Hamster models and led to much faster recovery. Its mode of action has been elucidated by solving the atomic structure of its interaction with SARS-CoV-2. The antibody competes with ACE2 binding by blocking a large area of the SARS-CoV-2 spike protein.

Regulatory T cells engineered with a novel insulin-specific chimeric antigen receptor as a candidate immunotherapy for type 1 diabetes.

Adoptive immunotherapy with ex vivo expanded, polyspecific regulatory T cells (Tregs) is a promising treatment for graft-versus-host disease. Animal transplantation models used by us and others have demonstrated that the adoptive transfer of allospecific Tregs offers greater protection from graft rejection than that of polyclonal Tregs. This finding is in contrast to those of autoimmune models, where adoptive transfer of polyspecific Tregs had very limited effects, while antigen-specific Tregs were promising. However, antigen-specific Tregs in autoimmunity cannot be isolated in sufficient numbers. Chimeric antigen receptors (CARs) can modify T cells and redirect their specificity toward needed antigens and are currently clinically used in leukemia patients. A major benefit of CAR technology is its "off-the-shelf" usability in a translational setting in contrast to major histocompatibility complex (MHC)-restricted T cell receptors. We used CAR technology to redirect T cell specificity toward insulin and redirect T effector cells (Teffs) to Tregs by Foxp3 transduction. Our data demonstrate that our converted, insulin-specific CAR Tregs (cTregs) were functional stable, suppressive and long-lived in vivo. This is a proof of concept for both redirection of T cell specificity and conversion of Teffs to cTregs.

Designing Human Antibodies by Phage Display.

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 strategies, including the adjustment of kinetic parameters.

Selection of Recombinant Human Antibodies.

Since the development of therapeutic antibodies the demand of recombinant human antibodies is steadily increasing. Traditionally, therapeutic antibodies were generated by immunization of rat or mice, the generation of hybridoma clones, cloning of the antibody genes and subsequent humanization and engineering of the lead candidates. In the last few years, techniques were developed that use transgenic animals with a human antibody gene repertoire. Here, modern recombinant DNA technologies can be combined with well established immunization and hybridoma technologies to generate already affinity maturated human antibodies. An alternative are in vitro technologies which enabled the generation of fully human antibodies from antibody gene libraries that even exceed the human antibody repertoire. Specific antibodies can be isolated from these libraries in a very short time and therefore reduce the development time of an antibody drug at a very early stage.In this review, we describe different technologies that are currently used for the in vitro and in vivo generation of human antibodies.

Generation of Recombinant Antibodies Against Toxins and Viruses by Phage Display for Diagnostics and Therapy.

Antibody phage display is an in vitro technology to generate recombinant antibodies. In particular for pathogens like viruses or toxins, antibody phage display is an alternative to hybridoma technology, since it circumvents the limitations of the immune system. Phage display allows the generation of human antibodies from naive antibody gene libraries when either immunized patients are not available or immunization is not ethically feasible. This technology also allows the construction of immune libraries to select in vivo affinity matured antibodies if immunized patients or animals are available.In this review, we describe the generation of human and human-like antibodies from naive antibody gene libraries and antibodies from immune antibody gene libraries. Furthermore, we give an overview about phage display derived recombinant antibodies against viruses and toxins for diagnostics and therapy.

Structural differences of amyloid-ß 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 ß-amyloid (Aß). During the underlying fibrillogenic pathway, starting from a surplus of soluble Aß and leading to mature fibrils, multiple conformations of this peptide appear, including oligomers of various shapes and sizes. To further investigate the fibrillization of ß-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 Aß. RESULTS: Here we report the generation and characterization of a variety of amyloid-ß specific human and human-like antibodies. Distinct fractions of monomers and oligomers of various sizes were separated by size exclusion chromatography (SEC) from Aß42 peptides. These antigens were used for the generation of two Aß42 specific immune scFv phage display libraries from macaque (Macaca fascicularis). Screening of these libraries as well as two naïve human phage display libraries resulted in multiple unique binders specific for amyloid-ß. Three of the obtained antibodies target the N-terminal part of Aß42 although with varying epitopes, while another scFv binds to the α-helical central region of the peptide. The affinities of the antibodies to various Aß42 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 ß-amyloid scFvs that are capable of accurately discriminating between minute conformational differences.

Generation and analysis of the improved human HAL9/10 antibody phage display libraries.

BACKGROUND: Antibody phage display is a proven key technology that allows the generation of human antibodies for diagnostics and therapy. From naive antibody gene libraries - in theory - antibodies against any target can be selected. Here we describe the design, construction and characterization of an optimized antibody phage display library. RESULTS: The naive antibody gene libraries HAL9 and HAL10, with a combined theoretical diversity of 1.5×10(10) independent clones, were constructed from 98 healthy donors using improved phage display vectors. In detail, most common phagemids employed for antibody phage display are using a combined His/Myc tag for detection and purification. We show that changing the tag order to Myc/His improved the production of soluble antibodies, but did not affect antibody phage display. For several published antibody libraries, the selected number of kappa scFvs were lower compared to lambda scFvs, probably due to a lower kappa scFv or Fab expression rate. Deletion of a phenylalanine at the end of the CL linker sequence in our new phagemid design increased scFv production rate and frequency of selected kappa antibodies significantly. The HAL libraries and 834 antibodies selected against 121 targets were analyzed regarding the used germline V-genes, used V-gene combinations and CDR-H3/-L3 length and composition. The amino acid diversity and distribution in the CDR-H3 of the initial library was retrieved in the CDR-H3 of selected antibodies showing that all CDR-H3 amino acids occurring in the human antibody repertoire can be functionally used and is not biased by E. coli expression or phage selection. Further, the data underline the importance of CDR length variations. CONCLUSION: The highly diverse universal antibody gene libraries HAL9/10 were constructed using an optimized scFv phagemid vector design. Analysis of selected antibodies revealed that the complete amino acid diversity in the CDR-H3 was also found in selected scFvs showing the functionality of the naive CDR-H3 diversity.

Application of M13 phage display for identifying immunogenic proteins from tick (Ixodes scapularis) saliva.

BACKGROUND: Ticks act as vectors for a large number of different pathogens, perhaps most notably Borrelia burgdorferi, the causative agent of Lyme disease. The most prominent tick vector in the United States is the blacklegged tick, Ixodes scapularis. Tick bites are of special public health concern since there are no vaccines available against most tick-transmitted pathogens. Based on the observation that certain non-natural host animals such as guinea pigs or humans can develop adaptive immune responses to tick bites, anti-tick vaccination is a potential approach to tackle health risks associated with tick bites. RESULTS: The aim of this study was to use an oligopeptide phage display strategy to identify immunogenic salivary gland proteins from I. scapularis that are recognized by human immune sera. Oligopeptide libraries were generated from salivary gland mRNA of 18 h fed nymphal I. scapularis. Eight immunogenic oligopeptides were selected using human immune sera. Three selected immunogenic oligopeptides were cloned and produced as recombinant proteins. The immunogenic character of an identified metalloprotease (MP1) was validated with human sera. This enzyme has been described previously and was hypothesized as immunogenic which was confirmed in this study. Interestingly, it also has close homologs in other Ixodes species. CONCLUSION: An immunogenic protein of I. scapularis was identified by oligopeptide phage display. MP1 is a potential candidate for vaccine development.

High level transient production of recombinant antibodies and antibody fusion proteins in HEK293 cells.

BACKGROUND: The demand of monospecific high affinity binding reagents, particularly monoclonal antibodies, has been steadily increasing over the last years. Enhanced throughput of antibody generation has been addressed by optimizing in vitro selection using phage display which moved the major bottleneck to the production and purification of recombinant antibodies in an end-user friendly format. Single chain (sc)Fv antibody fragments require additional tags for detection and are not as suitable as immunoglobulins (Ig)G in many immunoassays. In contrast, the bivalent scFv-Fc antibody format shares many properties with IgG and has a very high application compatibility. RESULTS: In this study transient expression of scFv-Fc antibodies in human embryonic kidney (HEK) 293 cells was optimized. Production levels of 10-20 mg/L scFv-Fc antibody were achieved in adherent HEK293T cells. Employment of HEK293-6E suspension cells expressing a truncated variant of the Epstein Barr virus (EBV) nuclear antigen (EBNA) 1 in combination with production under serum free conditions increased the volumetric yield up to 10-fold to more than 140 mg/L scFv-Fc antibody. After vector optimization and process optimization the yield of an scFv-Fc antibody and a cytotoxic antibody-RNase fusion protein further increased 3-4-fold to more than 450 mg/L. Finally, an entirely new mammalian expression vector was constructed for single step in frame cloning of scFv genes from antibody phage display libraries. Transient expression of more than 20 different scFv-Fc antibodies resulted in volumetric yields of up to 600 mg/L and 400 mg/L in average. CONCLUSION: Transient production of recombinant scFv-Fc antibodies in HEK293-6E in combination with optimized vectors and fed batch shake flasks cultivation is efficient and robust, and integrates well into a high-throughput recombinant antibody generation pipeline.