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

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