In a recent study published on the bioRxiv* preprint server, an international team of researchers took advantage of the human light chain apoferritin protomer-derived multiaffinity antibody platform (Multabody, MB) for the generation of broad SARS-CoV and powerful -2 (severe acute respiratory syndrome coronavirus 2)-neutralizing antibodies (Abs).
The continued emergence of worrisome SARS-CoV-2 (COV) variants has threatened the efficacy of anti-SARS-CoV-2 therapeutic monoclonal antibodies (mAbs). The authors of the study have previously developed the MB platform to improve the neutralization capacity of anti-SARS-CoV-2 and anti-human immunodeficiency virus 1 (HIV-1). They further reported that increased Ab affinity could be combined with multiple specificity (multiple Ab fragments recognizing different epitopes) to enhance antigen recognition.
Study: Antibody avidity and multispecificity combine to confer protection against SARS-CoV-2 and resilience against viral escape. Image credit: Corona Borealis Studio / Shutterstock
About the study
In the present study, the researchers investigated whether the previously reported enhanced in vitro SARS-CoV-2 neutralization for the MB platform could be translated into in vivo protection at low doses. They also investigated whether MBs could recover the loss of neutralization capacity observed for conventionally used mAbs against SARS-CoV-2 VOCs and extend their range of action to other viruses.
The tri-specific MB was assembled at atom-level resolution and examined by cryo-electron microscopy (cryo-EM) analysis. The ability of MB to provide immune protection in vivo was assessed by generating an IgG cocktail corresponding to MB that includes IgG4 Fc mutations (F234A, S228P, G237A, P238S, L235A) to eliminate binding to Fc-gamma receptors ( FcgRs). Biolayer interferometry (BLI) experiments and ADCP (Ab-dependent cell-mediated phagocytosis) experiments were performed.
Mice expressing human angiotensin-converting enzyme 2 (hACE2) and hFcRn were treated with FcgR-binding-deficient IgG4 and MB molecules and challenged intranasally with SARS-CoV-2. Lung samples were obtained from mice to determine pulmonary viral titers based on cytopathic effects (CPE) and quantitative reverse transcription-polymerase chain reaction (qRT-PCR) analysis.
To investigate whether the neutralizing potency of the mAbs could be further improved, monospecific MBs were expressed and their neutralization amplitude and capacity compared with their corresponding mAbs against wild-type (WT) SARS-CoV-2 strain and the VOCs Alpha, Beta, Gamma, Delta and Omicron BA.1.
Pseudovirus (PsV) neutralization assays and sarbecovirus neuralization assays were performed to determine neutralization titers. Furthermore, the molecular binding mechanisms of mAb 80 were explored.
The tri-specific 2-7-10-40-11-11 molecular coverage was assessed based on the individual and combined RBD buried surface area (BSA) of the Ab specificities showing their three-dimensional (3D) structures. Human embryonic kidney (HEK) 293T-ACE2 cells and Vero E6 cells were used for cell culture experiments and genes encoding fusions of human apoferritin, binding regions to fragment antigen (Fabs), fragment crystallizable regions (Fcs) and IgG. HEK 293F cells. Protein fractions were purified by chromatography and flow cytometry (FC) analysis was performed.
results
The cores of Ab-like molecules were homogeneous. Improvements in MB neutralization potency translated into improved in vivo immune protection at doses 30-fold lower than the corresponding IgGs. MBs potently neutralized SARS-CoV-2 variants by utilizing combined avidity, and several mAbs could be fused to one MB molecule (with 62 contact residues) to neutralize sarbecoviruses other than SARS-CoV-2 .
The MB apoferritin scaffold resembled the human apoferritin light chain. The mean maximal inhibitory concentration (IC50) value of tri-specific MB (TMB) was 0.0002 μg/mL, 1000 times greater than the IgG cocktail. IgG4 Ab cocktail and TMB bound murine and human FcRn, but not FcgR. TMB conferred significantly greater protection (60% survival) than the IgG4 Ab cocktail.
Five mAbs (80, 52, 80, 11-11, 10-40, and 2-36) showed 100% amplitude with an IC50 cutoff value of five μg/mL. However, when using an IC50 cutoff value of 0.01 μg/mL, only 11-11 and 10-40 neutralized two VOCs. Conversely, when used as monospecific MBs, three mAb specificities (2–7, 80, and 52) achieved 100% neutralization amplitude using the same cutoff value.
mAb 80 inhibited SARS-CoV-2 by preventing the interaction of the receptor-binding motif (RBM) with ACE2, and the heavy chain of the mAb and residues T478 and S477 were mainly responsible for host-viral interactions . The residues were mutated in VOC, reducing Ab binding, which was increased by MB. Similarly, the mutations reduced neutralization of SARS-CoV-2 VOC and other sarbecoviruses by mAbs 2-7, 10-40, and 11-11 incorporated into the trispecific molecule, which was restored by additional BSA coverage high-MB
Overall, the findings of the study showed that by targeting three fully overlapping functional RBD epitopes and through avidity neutralization potency gains, the 2-7-10-40-11-11 MB tri- specific provided a proof of concept for a broad and resistant, and potent neutralization of pan-sarbecovirus as a molecule.
*Important news
bioRxiv publishes preliminary scientific reports that are not peer-reviewed and therefore should not be considered conclusive, guide clinical practice/health-related behavior, or be treated as established information.