Difference between revisions of "Part:BBa K4040019"

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   <li style="display: inline-block;"> [[File:T--NMU_China--allviewcr3022bindstoRBD.jpeg|thumb|none|600px|<b>Figure 2:</b> Negative-stain EM and cryo-EM analysis of SARS spike bound to CR3022 Fab. TA) Representative 2D nsEM class averages of the trimeric SARS-CoV spike glycoprotein complexed with three CR3022 Fabs. (B) Side and top view of the 3D reconstruction showing CR3022 Fabs bound to all 3 RBDs on the SARS-CoV spike. The SARS-CoV RBD-CR3022 complex from the crystal structure is fitted into the nsEM density with the RBD shown in pink and CR3022 Fab in blue. (C) Side views of the B-factor-sharpened cryo-EM maps (transparent gray surface representation) representing three different classes of SARS spike with CR3022 Fab with different RBD-Fab orientations. While four different classes were identified, only three classes are shown here because classes 2 and 4 are very similar . The RBD-Fab complex model is fit into the densities with the RBDs shown in pink and CR3022 Fabs represented in blue. The atomic model of the apo SARS-CoV spike (PDB 6ACD) is also fit into density with one RBD removed for clarity. The protomers are colored in purple, magenta and deep magenta. (D) Top view of the class 2 cryo-EM map depicting potential quaternary contacts between the RBD-bound Fab and the spike NTD in this conformation. In this RBD-Fab conformation, the Fab would clash with the “down” RBD of the adjacent protomer (magenta) and, therefore, the adjacent RBD can only exist in an “up” conformation. (E) A close-up view of the Fab-spike interface showing the superimposition of CR3022 Fab and adjacent RBD. The residues that can contribute to quaternary interactions between CR3022 light chain and the NTD in two of the four classes (2 and 4) are shown. Date are from <html><a href="#4">[4]</a></html>.]] </li>
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   <li style="display: inline-block;"> [[File:T--NMU_China--allviewcr3022bindstoRBD.jpeg|thumb|none|500px|<b>Figure 2:</b> Negative-stain EM and cryo-EM analysis of SARS spike bound to CR3022 Fab. TA) Representative 2D nsEM class averages of the trimeric SARS-CoV spike glycoprotein complexed with three CR3022 Fabs. (B) Side and top view of the 3D reconstruction showing CR3022 Fabs bound to all 3 RBDs on the SARS-CoV spike. The SARS-CoV RBD-CR3022 complex from the crystal structure is fitted into the nsEM density with the RBD shown in pink and CR3022 Fab in blue. (C) Side views of the B-factor-sharpened cryo-EM maps (transparent gray surface representation) representing three different classes of SARS spike with CR3022 Fab with different RBD-Fab orientations. While four different classes were identified, only three classes are shown here because classes 2 and 4 are very similar . The RBD-Fab complex model is fit into the densities with the RBDs shown in pink and CR3022 Fabs represented in blue. The atomic model of the apo SARS-CoV spike (PDB 6ACD) is also fit into density with one RBD removed for clarity. The protomers are colored in purple, magenta and deep magenta. (D) Top view of the class 2 cryo-EM map depicting potential quaternary contacts between the RBD-bound Fab and the spike NTD in this conformation. In this RBD-Fab conformation, the Fab would clash with the “down” RBD of the adjacent protomer (magenta) and, therefore, the adjacent RBD can only exist in an “up” conformation. (E) A close-up view of the Fab-spike interface showing the superimposition of CR3022 Fab and adjacent RBD. The residues that can contribute to quaternary interactions between CR3022 light chain and the NTD in two of the four classes (2 and 4) are shown. Date are from <html><a href="#4">[4]</a></html>.]] </li>
 
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Revision as of 02:46, 28 September 2021


CR3022 scFv Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 352
    Illegal NgoMIV site found at 688
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI.rc site found at 568

Usage and Biology

CR3022 is a SARS-CoV neutralizing antibody to a highly conserved epitope on the receptor-binding domain (RBD) on the spike protein that is able to cross-react with SARS-CoV-2. A single-chain variable fragment (scFv) is not actually a fragment of an antibody, but instead is a fusion protein of the variable regions of the heavy (VH) and light chains (VL) of immunoglobulins, connected with a short linker peptide of ten to about 25 amino acids. The linker is usually rich in glycine for flexibility, as well as serine or threonine for solubility, and can either connect the N-terminus of the VH with the C-terminus of the VL or vice versa. This protein retains the specificity of the original immunoglobulin, despite the removal of the constant regions and the introduction of the linker. These molecules were created to facilitate phage display, where it is highly convenient to express the antigen-binding domain as a single peptide. As an alternative, scFv can be created directly from subcloned heavy and light chains derived from a hybridoma. ScFvs have many uses, e.g., flow cytometry, immunohistochemistry, and as antigen-binding domains of artificial T cell receptors (chimeric antigen receptor). CR3022 scFv is an scFv protein derived from the antibody CR3022.

Experimental results

A Broad-spectrum neutralizing antibody

CR3022 was previously isolated from a SARS survivor and neutralizes SARS-CoV [1], CR3022 was recently found to also be a cross-reactive antibody that can bind to both SARS-CoV-2 and SARS-CoV [2]. Recent crystal structure demonstrated that CR3022 targets a highly conserved cryptic epitope on the receptor binding domain (RBD) of the S protein [3]. The CR3022 epitope is exposed only when the RBD is in the “up” but not the “down” conformation on the S protein. A few SARS-CoV-2 antibodies from COVID-19 patients have also recently been shown to target the CR3022 epitope, suggesting that it is an important site of vulnerability for the antibody response in SARS-CoV-2 infection. Out of 28 residues in the CR3022 epitope, 24 are conserved between SARS-CoV-2 and SARS-CoV, explaining the cross-reactive binding of CR3022. However, CR3022 has a higher affinity to SARS-CoV than to SARS-CoV-2 (>100-fold difference), and can neutralize SARS-CoV, but not SARS-CoV-2, in a live virus neutralization assay [3]. Therefore, CR3022 provides a good case study to probe antigenic variation between SARS-CoV-2 and SARS-CoV and the effects on antibody cross-neutralization.

  • Figure 1: Crystal structure of SARS-CoV receptor binding domain in complex with human antibody CR3022. The structure has been resolved [4] available in PDB with accession number 7JN5.

Binding to RBD on the spike protein

  • Figure 2: Negative-stain EM and cryo-EM analysis of SARS spike bound to CR3022 Fab. TA) Representative 2D nsEM class averages of the trimeric SARS-CoV spike glycoprotein complexed with three CR3022 Fabs. (B) Side and top view of the 3D reconstruction showing CR3022 Fabs bound to all 3 RBDs on the SARS-CoV spike. The SARS-CoV RBD-CR3022 complex from the crystal structure is fitted into the nsEM density with the RBD shown in pink and CR3022 Fab in blue. (C) Side views of the B-factor-sharpened cryo-EM maps (transparent gray surface representation) representing three different classes of SARS spike with CR3022 Fab with different RBD-Fab orientations. While four different classes were identified, only three classes are shown here because classes 2 and 4 are very similar . The RBD-Fab complex model is fit into the densities with the RBDs shown in pink and CR3022 Fabs represented in blue. The atomic model of the apo SARS-CoV spike (PDB 6ACD) is also fit into density with one RBD removed for clarity. The protomers are colored in purple, magenta and deep magenta. (D) Top view of the class 2 cryo-EM map depicting potential quaternary contacts between the RBD-bound Fab and the spike NTD in this conformation. In this RBD-Fab conformation, the Fab would clash with the “down” RBD of the adjacent protomer (magenta) and, therefore, the adjacent RBD can only exist in an “up” conformation. (E) A close-up view of the Fab-spike interface showing the superimposition of CR3022 Fab and adjacent RBD. The residues that can contribute to quaternary interactions between CR3022 light chain and the NTD in two of the four classes (2 and 4) are shown. Date are from [4].

Used for a CAR

References

Ordered List

  1. ter Meulen J, van den Brink EN, Poon LL, Marissen WE, Leung CS, Cox F, Cheung CY, Bakker AQ, Bogaards JA, van Deventer E, Preiser W, Doerr HW, Chow VT, de Kruif J, Peiris JS, Goudsmit J. Human monoclonal antibody combination against SARS coronavirus: synergy and coverage of escape mutants. PLoS Med. 2006 Jul;3(7):e237. doi: 10.1371/journal.pmed.0030237. PMID: 16796401; PMCID: PMC1483912.
  2. Tian X, Li C, Huang A, Xia S, Lu S, Shi Z, Lu L, Jiang S, Yang Z, Wu Y, Ying T. Potent binding of 2019 novel coronavirus spike protein by a SARS coronavirus-specific human monoclonal antibody. Emerg Microbes Infect. 2020 Feb 17;9(1):382-385. doi: 10.1080/22221751.2020.1729069. PMID: 32065055; PMCID: PMC7048180.
  3. Yuan M, Wu NC, Zhu X, Lee CD, So RTY, Lv H, Mok CKP, Wilson IA. A highly conserved cryptic epitope in the receptor binding domains of SARS-CoV-2 and SARS-CoV. Science. 2020 May 8;368(6491):630-633. doi: 10.1126/science.abb7269. Epub 2020 Apr 3. PMID: 32245784; PMCID: PMC7164391.
  4. Wu NC, Yuan M, Bangaru S, Huang D, Zhu X, Lee CD, Turner HL, Peng L, Yang L, Burton DR, Nemazee D, Ward AB, Wilson IA. A natural mutation between SARS-CoV-2 and SARS-CoV determines neutralization by a cross-reactive antibody. PLoS Pathog. 2020 Dec 4;16(12):e1009089. doi: 10.1371/journal.ppat.1009089. PMID: 33275640; PMCID: PMC7744049.