Entwicklung neutralisierender und nicht-neutralisierender Antikörper gegen bekannte und neue Epitope auf Clostridioides difficile Toxin B

In this study, antibody phage display technology was used to generate a broad panel of fully human monoclonal antibodies that target TcdB, one of the main virulence factors of Clostridioides difficile and target of new therapeutic approaches. TcdB has a complex domain structure and intoxicates cells via a multistep mechanism, thus offering several weak points which can be addressed with antibodies. Applying different panning strategies against either full TcdB, truncated variants or single domains, 36 unique scFvs were generated of which 31 were further characterized after conversion into the bivalent scFv-Fc format. ELISA and Immunoblot analysis proved TcdB binding and domain specificity of these 31 antibodies analyzed, and confirmed the selection of antibodies with diverse binding characteristics. Using a cell-based in vitro assay, all antibodies were screened for neutralization of TcdB induced cell rounding. Here two antibodies efficiently neutralized TcdB. To test whether in vitro neutralization could be further improved by targeting different domains of TcdB at the same time, antibody combinations were tested in in vitro neutralization of TcdB induced cell rounding. Unfortunately, for the combinations tested in this study, the improvement of neutralization observed was rather of additive than synergistic nature. To gain more information about the binding sites of the antibodies two independent approaches of epitope mapping were followed. With the combination of Peptide array and TcdB-fragment phage display, novel and already described epitopes on TcdB could be identified. By correlating these epitopes with the in vitro neutralization efficacy, a new epitope within the glucosyltransferase domain of TcdB was identified which conveys neutralization. Thus, this study provides valuable information for further development of therapeutics against TcdB. Also, the two potent neutralizing antibodies might be candidates for further preclinical development. Future therapeutic use of these antibodies, preferably as antibody combination with antibodies targeting different domains or epitopes, is easily conceivable due to the human origin of these antibodies.In dieser Studie wurde die Antikörper-Phagen-Display-Technologie zur Generierung vollständig humaner monoklonaler Antikörper gegen TcdB, einen der wichtigsten Virulenzfaktoren von Clostridioides difficile, eingesetzt. TcdB hat eine komplexe Domänenstruktur und vergiftet Zellen über einen mehrschrittigen Mechanismus. Somit umfasst es mehrere Regionen, an denen Antikörper angreifen können um das Toxin zu neutralisieren. In mehreren Bio-Pannings, entweder gegen das vollständige TcdB, verkürzte Varianten oder einzelne Domänen, wurden 36 einzigartige scFvs generiert, von denen 31 nach Konvertierung in das bivalente scFv-Fc-Format weiter charakterisiert wurden. Mittels Antigen-ELISA und Immunoblot konnte die TcdB-Bindung dieser 31 Antikörper validiert und die Domänenspezifität bestimmt werden. Über einen zellbasierten in vitro Assay wurden die Antikörper auf Neutralisierung der durch TcdB-induzierten Zellrundung untersucht. Zwei der Antikörper neutralisieren TcdB besonders effizient. Durch die simultane Adressierung verschiedener Domänen von TcdB mit Antikörperkombinationen konnte die in vitro Neutralisierung der durch TcdB-induzierten Zellrundung weiter verbessert werden. Die beobachtete Verbesserung der Neutralisierung war allerdings eher auf additive als auf synergistische Effekte zurückzuführen. Des Weiteren wurden zwei unabhängige Ansätze zur Epitop Kartierung der Antikörper verfolgt. Mit der Kombination von Peptid-Array und TcdB-Fragment-Phagen-Display konnten neue sowie bereits beschriebene Epitope auf TcdB identifiziert werden. Durch Korrelation der Epitope mit der Neutralisierungseffizienz der gegen sie gerichteten Antikörper, konnte ein neues Epitop innerhalb der Glucosyltransferase-Domäne identifiziert werden welches Neutralisierung vermittelt. Somit liefert diese Studie wertvolle Informationen für die weitere Entwicklung von Therapeutika gegen TcdB. Auch die beiden potenten neutralisierenden Antikörper sind mögliche Kandidaten für eine weitere präklinische Entwicklung. Ein therapeutischer Einsatz dieser Antikörper, vorzugsweise als Antikörper-Kombination mit Antikörpern, die gegen andere Domänen oder Epitope gerichtet sind, ist aufgrund des menschlichen Ursprungs dieser Antikörper gut vorstellbar

The Conserved Cys-2232 in Clostridioides difficile Toxin B Modulates Receptor Binding

Clostridioides difficile toxins TcdA and TcdB are large clostridial glucosyltransferases which are the main pathogenicity factors in C. difficile-associated diseases. Four highly conserved cysteines are present in all large clostridial glucosyltransferases. In this study we focused on the conserved cysteine 2232 within the combined repetitive oligopeptide domain of TcdB from reference strain VPI10463 (clade I). Cysteine 2232 is not present in TcdB from hypervirulent strain R20291 (clade II), where a tyrosine is found instead. Replacement of cysteine 2232 by tyrosine in TcdBV PI10463 reduced binding to the soluble fragments of the two known TcdB receptors, frizzled-2 (FZD2) and poliovirus receptor-like protein-3/nectin-3 (PVRL3). In line with this, TcdBR20291 showed weak binding to PVRL3 in pull-down assays which was increased when tyrosine 2232 was exchanged for cysteine. Surprisingly, we did not observe binding of TcdBR20291 to FZD2, indicating that this receptor is less important for this toxinotype. Competition assay with the receptor binding fragments (aa 1101–1836) of TcdBV PI10463 and TcdBR20291, as well as antibodies newly developed by antibody phage display, revealed different characteristics of the yet poorly described delivery domain of TcdB harboring the second receptor binding region. In summary, we found that conserved Cys-2232 in TcdB indirectly contributes to toxin–receptor interaction

The Binary Toxin CDT of Clostridium difficile as a Tool for Intracellular Delivery of Bacterial Glucosyltransferase Domains

Binary toxins are produced by several pathogenic bacteria. Examples are the C2 toxin from Clostridium botulinum, the iota toxin from Clostridium perfringens, and the CDT from Clostridium difficile. All these binary toxins have ADP-ribosyltransferases (ADPRT) as their enzymatically active component that modify monomeric actin in their target cells. The binary C2 toxin was intensively described as a tool for intracellular delivery of allogenic ADPRTs. Here, we firstly describe the binary toxin CDT from C. difficile as an effective tool for heterologous intracellular delivery. Even 60 kDa glucosyltransferase domains of large clostridial glucosyltransferases can be delivered into cells. The glucosyltransferase domains of five tested large clostridial glucosyltransferases were successfully introduced into cells as chimeric fusions to the CDTa adapter domain (CDTaN). Cell uptake was demonstrated by the analysis of cell morphology, cytoskeleton staining, and intracellular substrate glucosylation. The fusion toxins were functional only when the adapter domain of CDTa was N-terminally located, according to its native orientation. Thus, like other binary toxins, the CDTaN/b system can be used for standardized delivery systems not only for bacterial ADPRTs but also for a variety of bacterial glucosyltransferase domains

Development of Neutralizing and Non-neutralizing Antibodies Targeting Known and Novel Epitopes of TcdB of Clostridioides difficile.

Clostridioides difficile is the causative bacterium in 15-20% of all antibiotic associated diarrheas. The symptoms associated with C. difficile infection (CDI) are primarily induced by the two large exotoxins TcdA and TcdB. Both toxins enter target cells by receptor-mediated endocytosis. Although different toxin receptors have been identified, it is no valid therapeutic option to prevent receptor endocytosis. Therapeutics, such as neutralizing antibodies, directly targeting both toxins are in development. Interestingly, only the anti-TcdB antibody bezlotoxumab but not the anti-TcdA antibody actoxumab prevented recurrence of CDI in clinical trials. In this work, 31 human antibody fragments against TcdB were selected by antibody phage display from the human naive antibody gene libraries HAL9/10. These antibody fragments were further characterized by in vitro neutralization assays. The epitopes of the neutralizing and non-neutralizing antibody fragments were analyzed by domain mapping, TcdB fragment phage display, and peptide arrays, to identify neutralizing and non-neutralizing epitopes. A new neutralizing epitope within the glucosyltransferase domain of TcdB was identified, providing new insights into the relevance of different toxin regions in respect of neutralization and toxicity

Developing Recombinant Antibodies by Phage Display Against Infectious Diseases and Toxins for Diagnostics and Therapy

Antibodies are essential molecules for diagnosis and treatment of diseases caused by pathogens and their toxins. Antibodies were integrated in our medical repertoire against infectious diseases more than hundred years ago by using animal sera to treat tetanus and diphtheria. In these days, most developed therapeutic antibodies target cancer or autoimmune diseases. The COVID-19 pandemic was a reminder about the importance of antibodies for therapy against infectious diseases. While monoclonal antibodies could be generated by hybridoma technology since the 70ies of the former century, nowadays antibody phage display, among other display technologies, is robustly established to discover new human monoclonal antibodies. Phage display is an in vitro technology which confers the potential for generating antibodies from universal libraries against any conceivable molecule of sufficient size and omits the limitations of the immune systems. If convalescent patients or immunized/infected animals are available, it is possible to construct immune phage display libraries to select in vivo affinity-matured antibodies. A further advantage is the availability of the DNA sequence encoding the phage displayed antibody fragment, which is packaged in the phage particles. Therefore, the selected antibody fragments can be rapidly further engineered in any needed antibody format according to the requirements of the final application. In this review, we present an overview of phage display derived recombinant antibodies against bacterial, viral and eukaryotic pathogens, as well as microbial toxins, intended for diagnostic and therapeutic applications

A SARS-CoV-2 neutralizing antibody selected from COVID-19 patients binds to the ACE2-RBD interface and is tolerant to most known RBD mutations

The novel betacoronavirus severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) causes a form of severe pneumonia disease called coronavirus disease 2019 (COVID-19). To develop human neutralizing anti-SARS-CoV-2 antibodies, antibody gene libraries from convalescent COVID-19 patients were constructed and recombinant antibody fragments (scFv) against the receptor-binding domain (RBD) of the spike protein were selected by phage display. The antibody STE90-C11 shows a subnanometer IC50 in a plaque-based live SARS-CoV-2 neutralization assay. The in vivo efficacy of the antibody is demonstrated in the Syrian hamster and in the human angiotensin-converting enzyme 2 (hACE2) mice model. The crystal structure of STE90-C11 Fab in complex with SARS-CoV-2-RBD is solved at 2.0 Å resolution showing that the antibody binds at the same region as ACE2 to RBD. The binding and inhibition of STE90-C11 is not blocked by many known emerging RBD mutations. STE90-C11-derived human IgG1 with FcγR-silenced Fc (COR-101) is undergoing Phase Ib/II clinical trials for the treatment of moderate to severe COVID-19

Expression of the Human Cytomegalovirus Pentamer Complex for vaccine use in a CHO system

Human cytomegalovirus (HCMV) causes significant disease worldwide. Multiple HCMV vaccines have been tested in man but only partial protection has been achieved. The HCMV gH/gL/UL128/UL130/UL131A complex (Pentamer) is the main target of neutralizing antibodies in HCMV seropositive individuals and immunization with Pentamer raises high titers of neutralizing antibodies in small animals and non-human primates (NHP). Thus, Pentamer is a promising candidate for a future HCMV vaccine. Development of a Pentamer-based subunit vaccine requires expression of high amounts of a functional and stable complex. We describe here the development of a mammalian expression system for large scale Pentamer production. Several approaches comprising three different CHO-originated cell lines and multiple vector as well as selection strategies were tested. Stable cell pools expressed the HCMV Pentamer at a titer of approximately 60 mg/L at laboratory scale. A FACS-based single cell sorting approach allowed selection of a high expressing clone producing Pentamer at levels of approximately 400 mg/L in a laboratory scale fed-batch culture. Expression in a 50L bioreactor led to the production of HCMV Pentamer at comparable titers indicating the feasibility of further scale-up to production scale. The CHO produced HCMV Pentamer complex bound to a panel of human neutralizing antibodies and raised potently neutralizing immune response in mice. Thus, we have generated an expression system for the large scale production of functional HCMV Pentamer at high titers suitable for future subunit vaccine production

SARS-CoV-2 neutralizing human recombinant antibodies selected from pre-pandemic healthy donors binding at RBD-ACE2 interface.

COVID-19 is a severe acute respiratory disease caused by SARS-CoV-2, a new recently emerged sarbecovirus. This virus uses the human ACE2 enzyme as receptor for cell entry, recognizing it with the receptor binding domain (RBD) of the S1 subunit of the viral spike protein. We present the use of phage display to select anti-SARS-CoV-2 spike antibodies from the human naïve antibody gene libraries HAL9/10 and subsequent identification of 309 unique fully human antibodies against S1. 17 antibodies are binding to the RBD, showing inhibition of spike binding to cells expressing ACE2 as scFv-Fc and neutralize active SARS-CoV-2 virus infection of VeroE6 cells. The antibody STE73-2E9 is showing neutralization of active SARS-CoV-2 as IgG and is binding to the ACE2-RBD interface. Thus, universal libraries from healthy human donors offer the advantage that antibodies can be generated quickly and independent from the availability of material from recovering patients in a pandemic situation

A SARS-CoV-2 neutralizing antibody selected from COVID-19 patients binds to the ACE2-RBD interface and is tolerant to most known RBD mutations.

The novel betacoronavirus severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) causes a form of severe pneumonia disease called coronavirus disease 2019 (COVID-19). To develop human neutralizing anti-SARS-CoV-2 antibodies, antibody gene libraries from convalescent COVID-19 patients were constructed and recombinant antibody fragments (scFv) against the receptor-binding domain (RBD) of the spike protein were selected by phage display. The antibody STE90-C11 shows a subnanometer IC50 in a plaque-based live SARS-CoV-2 neutralization assay. The in vivo efficacy of the antibody is demonstrated in the Syrian hamster and in the human angiotensin-converting enzyme 2 (hACE2) mice model. The crystal structure of STE90-C11 Fab in complex with SARS-CoV-2-RBD is solved at 2.0 Å resolution showing that the antibody binds at the same region as ACE2 to RBD. The binding and inhibition of STE90-C11 is not blocked by many known emerging RBD mutations. STE90-C11-derived human IgG1 with FcγR-silenced Fc (COR-101) is undergoing Phase Ib/II clinical trials for the treatment of moderate to severe COVID-19