CRRSP33 Antibody

What is CR3022 antibody and where was it originally isolated from?

CR3022 is a monoclonal antibody originally isolated from a convalescent SARS patient during the SARS-CoV-1 outbreak . It gained renewed research interest during the COVID-19 pandemic when it was found to cross-react with SARS-CoV-2 . This antibody targets the receptor-binding domain (RBD) of the spike protein, binding to a conserved region that does not overlap with the ACE2 receptor-binding surface . Its ability to recognize both SARS-CoV and SARS-CoV-2 makes it valuable for studying conserved epitopes across coronaviruses.

What binding affinity does CR3022 demonstrate toward SARS-CoV-2?

Biolayer interferometry measurements have determined that CR3022 Fab binds to the trimeric SARS-CoV-2 spike protein with a dissociation constant (Kd) of approximately 80 nM . This affinity is substantially weaker than its binding to SARS-CoV, despite recognizing conserved epitopes . The binding kinetics were determined through rigorous association and dissociation rate analyses in phosphate buffer (pH 7.4, 70 mM NaCl), with association measured for 10 minutes followed by 30 minutes of dissociation .

How does CR3022 binding compare between SARS-CoV and SARS-CoV-2?

Despite binding to both viruses, CR3022 exhibits "substantially weaker binding" to SARS-CoV-2 than to SARS-CoV . This difference is particularly significant considering that SARS-CoV and SARS-CoV-2 share approximately 77% amino acid identity in their spike proteins . The differential binding characteristics likely contribute to the observation that while CR3022 can neutralize SARS-CoV, plaque reduction neutralization assays clearly demonstrate it cannot neutralize SARS-CoV-2 .

What structural changes occur when CR3022 binds to the SARS-CoV-2 spike protein?

Cryo-electron microscopy studies at 3.7 Å resolution have revealed that CR3022 binding induces dramatic conformational changes in the SARS-CoV-2 spike glycoprotein . The antibody binds to an epitope on the RBD that is not accessible in either the open or closed forms of the trimeric spike . Upon binding, CR3022 triggers significant rearrangements in the S1 domain that ultimately result in dissociation of the spike protein . This conformational disruption leads to the S1 domain assuming a new conformation that is incompatible with the trimeric structure of the pre-fusion spike .

What techniques were most valuable in determining CR3022's binding mechanism?

Multiple complementary techniques provided critical insights into CR3022's interaction with SARS-CoV-2:

• Cryo-Electron Microscopy (Cryo-EM): Achieved 3.7 Å resolution, enabling detailed visualization of the binding interface and conformational changes

• X-ray Crystallography: Used to image antibodies attached to their target sites on SARS-CoV-2, providing atomic-structure details

• Biolayer Interferometry: Quantified binding kinetics and determined dissociation constants

• Plaque Reduction Neutralization Assays: Established that despite binding, CR3022 does not neutralize SARS-CoV-2

The high resolution in cryo-EM studies was partly enabled by the formation of an adventitious but stable dimer of two CR3022/S1 complexes over time .

Why does CR3022 fail to neutralize SARS-CoV-2 despite binding to its spike protein?

Despite binding with reasonable affinity, CR3022 does not neutralize SARS-CoV-2 . Several factors explain this phenomenon:

• The epitope recognized by CR3022 does not overlap with the ACE2 receptor-binding surface, so it doesn't directly block virus-receptor interactions

• CR3022 binds to a cryptic epitope that is not readily accessible in the native conformation of the spike

• While CR3022 induces spike dissociation, this appears to occur at a stage where structural disruption cannot prevent infection

• The antibody may bind as the spike protein samples different conformational states, a phenomenon similar to that observed with hidden epitopes on influenza hemagglutinin

This highlights the complexity of antibody-mediated neutralization, demonstrating that binding alone is insufficient for neutralization.

How can CR3022 binding data inform vaccine design against coronaviruses?

The detailed structural understanding of CR3022 binding provides valuable insights for vaccine development:

• Identifying conserved epitopes across coronaviruses helps design broadly protective vaccines

• Understanding the structural basis of antibody-antigen interactions reveals potential immunogen designs that could elicit similar cross-reactive antibodies

• The atomic-resolution data of binding interfaces can guide structure-based vaccine design

Research on potent neutralizing antibodies has shown that many effective antibodies against SARS-CoV-2 share common features, such as being encoded by the IGHV3-53 gene . These antibodies are generally present in small numbers in healthy people's blood, suggesting that vaccines boosting these naturally occurring antibodies could provide effective protection .

What protein constructs are optimal for studying CR3022 interactions?

For structural and binding studies, researchers have successfully used the following constructs:

• SARS-CoV-2 Spike Ectodomain: A stabilized trimeric version (residues 1-1208 of YP_009724390.1) with modifications including:

  • "FUR 2P" stabilizing mutations (R682S, R685S, K986P, V987P)

  • Removal of the polybasic cleavage site between S1 and S2

  • Addition of a μ-phosphatase signal peptide, TEV-cleavage site, foldon trimerization motif, and hexahistidine tag

• CR3022 Fab: Expressed in mammalian cells for binding and structural studies

These optimized constructs facilitate high-quality structural and biochemical analyses.

What are the key methodological considerations for analyzing CR3022-spike interactions?

When designing experiments to study CR3022:

• Buffer Conditions: pH 7.4, 70 mM NaCl phosphate buffer has been successfully used for binding studies

• Protein Immobilization: For biolayer interferometry, spike glycoprotein at 20–40 μg/mL can be immobilized for ~40 minutes on NiNTA sensors

• Data Analysis: Association phases should be analyzed as single exponential functions, with plots of observed rate versus antibody concentration to determine association and dissociation rate constants

• Model Building: Begin with high-resolution density maps to build core regions based on pre-existing structures, followed by the addition of less well-ordered regions through rigid body refinement

How does CR3022 compare to other coronavirus-targeting antibodies?

While CR3022 binds to SARS-CoV-2 but fails to neutralize it, other antibodies show different characteristics:

• Scientists have discovered a subset of antibodies that are particularly powerful at neutralizing SARS-CoV-2

• Many potent neutralizing antibodies are encoded by the IGHV3-53 gene

• Some antibodies, like those discovered by Scripps Research, provide "powerful protection against SARS-CoV-2" when tested in animals and human cell cultures

Understanding these differences helps researchers develop more effective therapeutic antibodies and vaccines.

What can we learn from comparing CR3022 with neutralizing antibodies?

The comparison between non-neutralizing antibodies like CR3022 and potent neutralizing antibodies provides several insights:

• Binding location is critical - antibodies targeting regions that directly interfere with receptor binding tend to be more neutralizing

• Conformational requirements play an important role - some antibodies may only bind to specific conformational states of the spike protein

• Binding affinity doesn't always correlate with neutralization potency

• Cross-reactivity between coronaviruses often involves conserved epitopes that may not be optimal neutralization targets