Development of an inhibiting antibody against equine interleukin 5 to treat insect bite hypersensitivity of horses

Insect bite hypersensitivity (IBH) is the most common allergic skin disease of horses. It is caused by insect bites of the Culicoides spp. which mediate a type I/IVb allergy with strong involvement of eosinophil cells. No specific treatment option is available so far. One concept could be the use of a therapeutic antibody targeting equine interleukin 5, the main activator and regulator of eosinophils. Therefore, antibodies were selected by phage display using the naïve human antibody gene libraries HAL9/10, tested in a cellular in vitro inhibition assay and subjected to an in vitro affinity maturation. In total, 28 antibodies were selected by phage display out of which eleven have been found to be inhibiting in the final format as chimeric immunoglobulin G with equine constant domains. The two most promising candidates were further improved by in vitro affinity maturation up to factor 2.5 regarding their binding activity and up to factor 2.0 regarding their inhibition effect. The final antibody named NOL226-2-D10 showed a strong inhibition of the interleukin 5 binding to its receptor (IC50 = 4 nM). Furthermore, a nanomolar binding activity (EC50 = 8.8 nM), stable behavior and satisfactory producibility were demonstrated. This antibody is an excellent candidate for in vivo studies for the treatment of equine IBH

Development of an inhibiting antibody against equine interleukin 5 to treat insect bite hypersensitivity of horses

Insect bite hypersensitivity (IBH) is the most common allergic skin disease of horses. It is caused by insect bites of the Culicoides spp. which mediate a type I/IVb allergy with strong involvement of eosinophil cells. No specific treatment option is available so far. One concept could be the use of a therapeutic antibody targeting equine interleukin 5, the main activator and regulator of eosinophils. Therefore, antibodies were selected by phage display using the naïve human antibody gene libraries HAL9/10, tested in a cellular in vitro inhibition assay and subjected to an in vitro affinity maturation. In total, 28 antibodies were selected by phage display out of which eleven have been found to be inhibiting in the final format as chimeric immunoglobulin G with equine constant domains. The two most promising candidates were further improved by in vitro affinity maturation up to factor 2.5 regarding their binding activity and up to factor 2.0 regarding their inhibition effect. The final antibody named NOL226-2-D10 showed a strong inhibition of the interleukin 5 binding to its receptor (IC50 = 4 nM). Furthermore, a nanomolar binding activity (EC50 = 8.8 nM), stable behavior and satisfactory producibility were demonstrated. This antibody is an excellent candidate for in vivo studies for the treatment of equine IBH

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

Entwicklung von inhibierenden Antikörpern gegen equines Interleukin-5 und equines Interleukin-31 zur Behandlung des Sommerekzems von Pferden

Insect bite hypersensitivity (IBH) is the most common allergic skin disease of horses. It is caused by insect bites of the Culicoides spp. which mediate a type I/IVb allergy with strong involvement of T helper 2 (TH2) and eosinophil cells. The TH2-derived proinflammatory cytokines equine (eq) interleukin (IL)-5 and eqIL-31 play a key role in the development of IBH by causing eosinophilia and allergic pruritus. No specific treatment option is available so far that can be applied long term without safety concerns. One concept could be the use of therapeutic antibodies targeting eqIL-5, the main activator and regulator of eosinophils, and eqIL-31, the itch-mediating cytokine. This concept contains the challenge of working with equine targets and equine antibodies while there is no monoclonal therapeutic antibody for horses available so far. Antibodies against eqIL-5 and eqIL-31 were selected with different phage display techniques using the naïve human antibody gene libraries HAL9/10. These antibodies were tested regarding their functionality in a self-established cellular in vitro inhibition assay. In total, 28 producible antibodies binding to eqIL-5 and 62 producible antibodies binding to eqIL-31 were selected. Hereof, eleven antibodies anti-eqIL-5 and five antibodies anti-eqIL-31 were found to be inhibiting in the final format as chimeric immunoglobulin G with equine constant domains. After analyzing the stability and specificity, two lead candidates against eqIL-5 (NOL48-1-D5 and NOL162-1-F5) and one lead candidate against eqIL-31 (NOL48-3-A2) were subjected to an in vitro affinity maturation. The affinity-matured antibodies targeting eqIL-5 had improved binding activities up to factor 2.5 and improved inhibition effects up to factor 2.0 in comparison to the parental antibodies. The binding activity and inhibition effect of the affinity-matured antibodies targeting eqIL-31 were even improved by a higher factor. Despite stability issues of several antibodies after in vitro affinity maturation, one final antibody candidate against eqIL-5 (NOL226-2-D10) was selected which fulfilled all requirements that were analyzed in this study: a strong inhibition effect (IC50 = 4 nM, in competitive assay IC50 = 9 nM), nanomolar binding activity to the target (EC50 = 8.8 nM), satisfactory producibility (70 mg/L in 250 mL production scale in transient expression system) and high stability. This antibody was applied in a first in vivo orientation study, consisting of three antibody-treated horses and three control horses. The antibody-treated horses received an antibody dose of 42.6 µg antibody/kg body weight on day 0 and day 14 of the study. Taking into account the analyzed parameters score sheet, anti-drug antibody (ADA) assay as well as blood eosinophil and neutrophil level, the antibody NOL226-2-D10 displayed a high safety profile and first positive hints regarding its efficacy. Thus, this treatment concept can be further developed with the aim of achieving a targeted and safe therapy for equine IBH and thereby contribute to the generation of therapeutic antibodies in the veterinary field.Das Sommerekzem ist die häufigste allergische Hauterkrankung bei Pferden. Es wird durch Stiche von Insekten der Gattung Culicoides verursacht, welche eine Typ I/IVb Allergie mit starker Beteiligung von TH2 und eosinophilen Zellen vermitteln. Die von TH2 Zellen ausgeschütteten proinflammatorischen Zytokine equines Interleukin (eqIL)-5 und eqIL-31 spielen eine Schlüsselrolle bei der Entwicklung des Sommerekzems, indem sie Eosinophilie und allergischen Juckreiz auslösen. Bisher gibt es keine spezifische Behandlungsmöglichkeit, die langfristig und ohne Sicherheitsbedenken angewendet werden kann. Ein Behandlungskonzept könnte die Verwendung therapeutischer Antikörper sein, die eqIL-5, den Hauptaktivator und -regulator der Eosinophilen, und eqIL-31, das Juckreiz vermittelnde Zytokin, adressieren. Dieses Konzept beinhaltet die Herausforderung des Arbeitens mit equinen Zielmolekülen und Antikörpern, während bisher kein monoklonaler therapeutischer Antikörper für Pferde zur Verfügung steht. Zur Umsetzung dieses Ansatzes wurden Antikörper gegen eqIL-5 und eqIL-31 mittels verschiedener Phagen Display Strategien unter Verwendung der naiven humanen Antikörper-Genbibliotheken HAL9/10 selektiert. Die Funktionalität der Antikörper wurde in einem selbst etablierten zellbasierten in vitro Inhibitionstest untersucht. Insgesamt wurden 28 produzierbare Antikörper gegen eqIL-5 und 62 produzierbare Antikörper gegen eqIL-31 selektiert. Hiervon erwiesen sich elf Antikörper gegen eqIL-5 und fünf Antikörper gegen eqIL-31 im finalen Format als chimäres Immunglobulin G mit konstanten equinen Domänen als inhibierend. Nach Bestätigung der Stabilität und Spezifität wurden zwei Kandidaten gegen eqIL-5 (NOL48-1-D5 und NOL162-1-F5) und ein Kandidat gegen eqIL-31 (NOL48-3-A2) einer in vitro Affinitätsreifung unterzogen. Die affinitätsgereiften Antikörper gegen eqIL-5 zeigten im Vergleich zu ihren jeweiligen parentalen Antikörpern eine bis zu Faktor 2,5 verbesserte Bindungsaktivität und eine bis zu Faktor 2,0 verbesserte Inhibierung. Die affinitätsgereiften Antikörper gegen eqIL-31 wurden sogar um einen höheren Faktor verbessert. Trotz Stabilitätsproblemen mehrere Antikörper nach der in vitro Affinitätsreifung, wurde ein finaler Antikörperkandidat gegen eqIL-5 selektiert, welcher alle in dieser Arbeit untersuchten Anforderungen erfüllte: Einen starken Inhibitionseffekt (IC50 = 4 nM, im kompetitiven Test IC50 = 9 nM), nanomolare Bindungsaktivität an das Zielmolekül (EC50 = 8,8 nM), zufriedenstellende Produzierbarkeit (70 mg/L im 250 mL Produktionsmaßstab im transienten Expressionssystem) und hohe Stabilität. Der Antikörper wurde in einer ersten in vivo Orientierungsstudie mit sechs Testpferden eingesetzt. Drei der Pferde erhielten am Tag 0 und am Tag 14 der Studie eine Dosis von 42,6 µg Antikörper/kg Körpergewicht. Unter Berücksichtigung der analysierten Parameter Score Sheet, Anti-Drug-Antikörper Assay sowie Eosinophilen- und Neutrophilenzahl im Blut wies der Antikörper NOL226-2-D10 ein hohes Sicherheitsprofil auf und zeigte erste positive Hinweise bezüglich seiner Wirksamkeit. So kann dieses Behandlungskonzept weiterentwickelt werden, mit dem Ziel einer gezielten und sicheren Therapie für das Sommerekzem der Pferde

Development of an inhibiting antibody against equine interleukin 5 to treat insect bite hypersensitivity of horses

Insect bite hypersensitivity (IBH) is the most common allergic skin disease of horses. It is caused by insect bites of the Culicoides spp. which mediate a type I/IVb allergy with strong involvement of eosinophil cells. No specific treatment option is available so far. One concept could be the use of a therapeutic antibody targeting equine interleukin 5, the main activator and regulator of eosinophils. Therefore, antibodies were selected by phage display using the naïve human antibody gene libraries HAL9/10, tested in a cellular in vitro inhibition assay and subjected to an in vitro affinity maturation. In total, 28 antibodies were selected by phage display out of which eleven have been found to be inhibiting in the final format as chimeric immunoglobulin G with equine constant domains. The two most promising candidates were further improved by in vitro affinity maturation up to factor 2.5 regarding their binding activity and up to factor 2.0 regarding their inhibition effect. The final antibody named NOL226-2-D10 showed a strong inhibition of the interleukin 5 binding to its receptor (IC50 = 4 nM). Furthermore, a nanomolar binding activity (EC50 = 8.8 nM), stable behavior and satisfactory producibility were demonstrated. This antibody is an excellent candidate for in vivo studies for the treatment of equine IBH

Baculovirus-free insect cell expression system for high yield antibody and antigen production.

Mammalian cells are the most commonly used production system for therapeutic antibodies. Protocols for the expression of recombinant antibodies in HEK293-6E cells in different antibody formats are described in detail. As model, antibodies against Kallikrein-related peptidase 7 (KLK7) were used. KLK7 is a key player in skin homeostasis and represents an emerging target for pharmacological interventions. Potent inhibitors can not only help to elucidate physiological and pathophysiological functions but also serve as a new archetype for the treatment of inflammatory skin disorders. Phage display-derived affinity-matured human anti-KLK7 antibodies were converted to scFv-Fc, IgG, and Fab formats and transiently produced in the mammalian HEK293-6E system. For the production of the corresponding antigen-KLK7-the baculovirus expression vector system (BEVS) and virus-free expression in Hi5 insect cells were used in a comparative approach. The target proteins were isolated by various chromatographic methods in a one- or multistep purification strategy. Ultimately, the interaction between anti-KLK7 and KLK7 was characterized using biolayer interferometry. Here, protocols for the expression of recombinant antibodies in different formats are presented and compared for their specific features. Furthermore, biolayer interferometry (BLI), a fast and high-throughput biophysical analytical technique to evaluate the kinetic binding constant and affinity constant of the different anti-KLK7 antibody formats against Kallikrein-related peptidase 7 is presented

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