Studies in Humanized Mice and Convalescent Humans Yield a SARS-CoV-2 Antibody Cocktail
Antibody therapies have become an important tool in treating diverse conditions such as autoimmune diseases, cardiovascular diseases, allergic conditions, infectious diseases, and cancers. Proven methods for developing such antibody therapies include using mice with genetically humanized immune systems and human B cells to produce fully human antibodies. These approaches have recently been used to develop antibody treatments for Ebola virus infections.
More recently, a team of researchers at Regeneron Pharmaceuticals used these two approaches in tandem to create a highly potent antibody cocktail against SARS-CoV-2.1 The goal of their work was to create a therapy with strong antiviral potency and enough antibody variability to minimize the escape of viral mutants. 1
SARS-CoV-2 Antibody Cocktail
The researchers focused on generating complementary pairs of highly potent antibodies against the receptor binding domain (RBD) of the SARS-CoV-2 spike protein. Targeting and disabling the viral spikes renders the virus unable to enter host cells. High-throughput workflows were conducted using both humanized mice and convalescent human B cells to generate monoclonal antibodies (mAb) against the SARS-CoV-2 viral spike.
Antibody Screening and Characterization
Blood samples were collected from the mice seven days after the last boost for determination of serum antibody titers. The antibodies were tittered using enzyme-linked immunosorbent assay (ELISA) plates which were read using a PerkinElmer VICTOR®.
Mice with the highest titers were used for antibody isolation. Thousands of RBD-binding mAbs were identified and screened for the following parameters:
- RBD binding affinity
- Epitope diversity
- Ability to block angiotensin-converting enzyme 2 (ACE2) receptor binding to RBD
- Ability to neutralize vesicular stomatitis virus (VSV)–based SARS-CoV-2 spike pseudoparticles [pVSV-SARS-CoV-2-S(mNeon)]
The screening identified more than 200 neutralizing antibodies with broad potency ranges. Several dozen were potent at picomolar (pM) levels. Forty of the antibodies were selected for further characterization based on their distinct sequences and strong neutralization potencies.
The variable heavy and light chains of the 40 selected antibodies underwent two rounds of PCR amplification and subsequent quantification using the PerkinElmer LabChip GX Touch HT Nucleic Acid Analyzer. The antibody repertoires were then sequenced using next generation sequencing workflows.
The antibodies underwent testing of their neutralization potency in VSV-based pseudoparticle assays. The nine strongest antibodies showed potencies ranging from 7 to 99 pM. Those nine antibodies were examined for cross-competition binding potential. The results identified several pairs of non-competing antibodies with pM neutralization potency.
The neutralizing potency of the four lead antibodies was confirmed by assessing their neutralization of:
- pVSV-SARS-CoV-2-S(mNeon) in the human lung epithelial Calu-3 cell line
- replicating VSV-SARS-CoV-2-S in Vero cells
- SARS-CoV-2 in VeroE6 cells
All four antibodies demonstrated similar strong neutralization potencies, and no combinations of the four leads exhibited synergistic neutralization activity.
Primary human cell bioassays were conducted to assess the ability of the four lead antibodies to mediate antibody-dependent cellular toxicity (ADCC) and antibody-dependent cellular phagocytosis (ADCP). The assays were conducted using natural killer (NK) cells and monocyte-derived phagocytes, using the PerkinElmer EnVision Multi-mode plate reader, and the PerkinElmer Opera Phenix High-Content Screening system. Each of the antibodies was found to successfully mediate both ADCC and ADCP.
Structural and epitope studies revealed that non-competing antibodies were binding at different epitopes on the RBD. Some contacted the RBD such that the ACE2 interface was overlapped. Others made contact from a different angle and at a different location in the RBD such that the antibody did not overlap the ACE2 interface.
These studies enabled the research team to identify highly potent individual antibodies that target the SARS-CoV-2 spike protein. The antibodies have distinct characteristics that complement each other and, combined, make for a promising antibody cocktail that is strongly potent against SARS-CoV-2 and minimizes the escape of virus mutants.
The sequential screening steps and analyses provided the group data that was used to continually sharpen the focus on the most promising antibodies. PerkinElmer’s VICTOR®, LabChip GX Touch HT Nucleic Acid Analyzer, EnVision Multi-mode plate reader, and Opera Phenix High-Content Screening system enabled the investigators on their path to a SARS-CoV-2 antibody cocktail.