CRK22 Antibody

What is CR3022 and what is its origin?

CR3022 is a human monoclonal antibody originally isolated from the blood of a convalescent SARS-CoV patient. It has gained significant research interest due to its ability to bind to both SARS-CoV and SARS-CoV-2, making it valuable for comparative coronavirus studies . This antibody was initially characterized for its ability to bind to mutated neutralization-resistant forms of the SARS-CoV S1-RBD, demonstrating potential utility against emerging variants .

What are the basic binding properties of CR3022 to coronaviruses?

CR3022 exhibits potent binding to SARS-CoV-2 receptor-binding domain (RBD) with a dissociation constant (KD) of 6.3 nM . It follows fast-on (kon of 1.84 × 10^5 Ms^-1) and slow-off (koff of 1.16 × 10^-3 s^-1) binding kinetics . Importantly, CR3022 demonstrates specificity for SARS-related coronaviruses, binding tightly to both SARS-CoV S protein and SARS-CoV-2 RBD and S protein, while showing no binding affinity to MERS S protein .

How does CR3022 compare to other SARS-CoV antibodies?

Unlike other SARS-CoV-specific neutralizing antibodies (such as m396 and CR3014) that target the ACE2 binding site of SARS-CoV, CR3022 recognizes a distinct epitope that does not overlap with the ACE2 binding site . This unique binding property allows CR3022 to cross-react with SARS-CoV-2, while antibodies like m396 and CR3014 failed to bind to the SARS-CoV-2 spike protein . Historically, CR3022 was able to completely neutralize both wild-type SARS-CoV and CR3014 escape viruses at a concentration of 23.5 μg/ml, and no escape variants could be generated with CR3022 .

What is the structural basis for CR3022's cross-reactivity?

CR3022 targets a highly conserved cryptic epitope that is distal from the receptor binding site on the spike protein . This conservation between SARS-CoV and SARS-CoV-2 enables cross-reactive binding. Structural modeling has revealed that this epitope is only exposed when at least two of the three spike proteins in the trimeric complex are in a conformation competent to bind the receptor . Interestingly, CR3022 binds more tightly to SARS-CoV because its epitope contains a glycan not present in SARS-CoV-2 .

What is CR3022's binding affinity to SARS-CoV-2 variants?

CR3022 exhibits excellent binding affinity to multiple SARS-CoV-2 variants, including the prominent UK (B.1.1.7) and South African (B.1.351/501Y.V2) variants . This consistent binding across variants suggests that CR3022 recognizes a conserved epitope that remains largely unchanged despite mutations in the spike protein, particularly in the RBD region . This property makes CR3022 valuable for studying evolutionary patterns in SARS-CoV-2 variants.

How do Fc effector functions contribute to CR3022's potential therapeutic utility?

Despite limited neutralization capacity against SARS-CoV-2, CR3022 demonstrates significant Fc-mediated effector functions . CR3022 bound to SARS-CoV-2 S protein can effectively bind to Fc gamma receptors 2a and 3a (FcγR2a and FcγR3a), enabling various immune responses . The antibody drives multiple effector functions including:

• Antibody-dependent cellular phagocytosis (ADCP)

• Antibody-dependent NK cell activation (ADNKA)

• Antibody-dependent complement deposition (ADCD)

• Antibody-dependent neutrophil phagocytosis (ADNP)

Notably, CR3022 maintains these effector functions even in the presence of ACE2, suggesting potential therapeutic value even when ACE2 is upregulated following infection .

How can CR3022 be utilized in diagnostic applications?

CR3022 can be employed in various diagnostic contexts due to its availability in multiple engineered formats. For COVID-19 diagnostic assays, researchers can utilize:

• Human immunoglobulin formats (IgG1, IgG2, IgG3, IgG4, IgA, IgM, and IgE) as serological controls

• ISOblend™ standard containing human IgG1, IgG3, IgM, and IgA formulated at equal amounts for use as a control or calibrator in COVID-19 diagnostic tests

• Species-specific formats (rabbit, mouse, cat, and ferret) for detection applications in various animal models

These diverse formats facilitate the development of highly specific and sensitive diagnostic assays for SARS-CoV-2 detection.

What methodologies can be used to assess CR3022 binding affinity?

Multiple complementary techniques can be employed to evaluate CR3022 binding characteristics:

• Enzyme-Linked Immunosorbent Assay (ELISA): Plates coated with 50 ng/well of antigen in PBS, incubated overnight at 4°C, followed by blocking with 5% BSA. Five-fold serially diluted antibody is added, followed by anti-human IgG1-HRP for detection. Signal is developed with TMB and read at 450 nm (reference 570 nm) .

• Surface Plasmon Resonance (SPR): Useful for determining binding kinetics (kon and koff rates) and affinity constants (KD). In previous studies, CR3022 demonstrated a KD of 6.3 nM to SARS-CoV-2 RBD .

• Bio-Layer Interferometry (BLI): Streptavidin biosensors labeled with biotinylated 2019-nCoV RBD can be used for competition assays to determine if CR3022 competes with ACE2 for binding .

What approaches can be used to improve CR3022's binding affinity to SARS-CoV-2?

Structure-based affinity maturation has successfully enhanced CR3022's binding to SARS-CoV-2 . The methodology involves:

• Structure-based molecular simulations to virtually mutate key residues in the complementarity-determining regions (CDRs)

• Selection of mutation sites based on mutation energy criteria

• Experimental verification through ELISA and SPR

• Assessment of autoimmune reactivity for safety

• Molecular dynamics simulation and binding free energy calculation (MM/PBSA)

Key mutations that significantly improved affinity include S103F/Y in HCDR-3 and S33R in LCDR-1, which together enhanced binding affinity more than ten-fold by creating additional hydrogen-bonding, hydrophobic interactions, and salt-bridges with SARS-CoV-2 RBD .

How should researchers interpret the neutralization versus Fc-mediated effects of CR3022?

While CR3022 shows limited neutralization capacity against SARS-CoV-2 even at high concentrations, it exhibits robust Fc-mediated effector functions . This apparent contradiction highlights the importance of looking beyond neutralization when evaluating therapeutic antibodies. Researchers should design experiments that assess both neutralization (through pseudovirus or live virus neutralization assays) and Fc-mediated functions (through ADCP, ADNKA, ADCD, and ADNP assays) . The ability of CR3022 to drive effector functions even in the presence of ACE2 suggests that antibodies targeting non-ACE2-competing epitopes might provide complementary mechanisms of protection, especially for clearance of infected cells .

What explains the differential binding of CR3022 to SARS-CoV versus SARS-CoV-2?

CR3022 binds more tightly to SARS-CoV than to SARS-CoV-2, limiting its therapeutic development against the latter . This differential binding is attributed to:

• The presence of a glycan in the CR3022 epitope of SARS-CoV that is absent in SARS-CoV-2

• Differences in the C-terminus residues of the RBD between SARS-CoV and SARS-CoV-2

Understanding these structural differences provides insights for rational antibody engineering to improve cross-reactivity. Researchers should consider these differences when designing experiments to compare antibody responses across coronaviruses.

How can CR3022 be used in combination therapy approaches?

Historical data indicates that CR3022 and CR3014 neutralized SARS-CoV synergistically by recognizing different epitopes on the RBD . This suggests a potential strategy for SARS-CoV-2:

• Identify antibodies that target the ACE2 binding site of SARS-CoV-2

• Combine with CR3022 that targets a non-overlapping epitope

• Assess synergistic effects through neutralization assays

• Evaluate the potential for preventing escape mutants

This combinatorial approach may provide broader protection against emerging variants and reduce the likelihood of escape mutants developing during treatment.

What methodologies are recommended for testing CR3022 against new coronavirus variants?

When evaluating CR3022 against new variants, researchers should implement a comprehensive testing strategy:

• Express recombinant spike proteins or RBDs containing variant mutations

• Perform binding assays (ELISA, SPR, BLI) to determine if affinity is altered

• Conduct neutralization assays with pseudoviruses carrying variant spike proteins

• Assess Fc-mediated effector functions against cells expressing variant spike proteins

• Compare results with other antibodies targeting different epitopes

This systematic approach provides a complete profile of how mutations affect different aspects of antibody functionality.

How can CR3022 contribute to understanding coronavirus evolution?

As a cross-reactive antibody binding to a conserved epitope, CR3022 offers unique insights into coronavirus evolution:

• Map conservation and divergence of epitopes across SARS-like coronaviruses

• Identify critical residues that remain unchanged during viral evolution

• Determine structural constraints that limit variation in certain epitopes

• Use CR3022 binding as a benchmark to classify new coronaviruses

These approaches can help predict which epitopes might remain conserved in future coronavirus variants or spillovers, informing broad-spectrum vaccine design strategies.