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