Part:BBa_K4040019

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 and neutralizing

While it is now known that SARS-CoV and SARS-CoV-2 differ in antigenicity despite relatively high sequence conservation, there is a paucity of understanding of the underlying molecular determinants of these antigenic changes and the structural consequences of these differences. While CR3022 cannot neutralize SARS-CoV-2 WT in almost all studies, it can neutralize the SARS-CoV-2 P384A mutant. The KD of CR3022 Fab to SARS-CoV-2 WT RBD is 68 nM, whereas to SARS-CoV-2 P384A RBD is 1 nM, indicating that the affinity threshold for neutralization of SARS-CoV-2 to this epitope is in the low nM range. However, despite having a low nM affinity to SARS-CoV-2 P384A RBD, CR3022 only weakly neutralizes SARS-CoV-2 P384A with an IC50 of 3.2 μg/ml and SARS-CoV with an IC50 of 5.2 μg/ml.

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

Chimeric antigen receptor T cells (also known as CAR T cells) are T cells that have been genetically engineered to produce an artificial T cell receptor for use in immunotherapy. Chimeric antigen receptors (CARs, also known as chimeric immunoreceptors, chimeric T cell receptors or artificial T cell receptors) are receptor proteins that have been engineered to give T cells the new ability to target a specific protein. The receptors are chimeric because they combine both antigen-binding and T cell activating functions into a single receptor. CAR-T cell therapy uses T cells engineered with CARs for cancer therapy. The premise of CAR-T immunotherapy is to modify T cells to recognize cancer cells in order to more effectively target and destroy them. Scientists harvest T cells from people, genetically alter them, then infuse the resulting CAR-T cells into patients to attack their tumors.CAR T cells can be both CD4+ and CD8+, with a 1-to-1 ratio of both cell types providing synergistic antitumor effects. CAR-T cells can be either derived from T cells in a patient's own blood (autologous) or derived from the T cells of another healthy donor (allogeneic). Once isolated from a person, these T cells are genetically engineered to express a specific CAR, which programs them to target an antigen that is present on the surface of tumors. For safety, CAR-T cells are engineered to be specific to an antigen expressed on a tumor that is not expressed on healthy cells. After CAR-T cells are infused into a patient, they act as a "living drug" against cancer cells.When they come in contact with their targeted antigen on a cell, CAR-T cells bind to it and become activated, then proceed to proliferate and become cytotoxic.CAR-T cells destroy cells through several mechanisms, including extensive stimulated cell proliferation, increasing the degree to which they are toxic to other living cells (cytotoxicity) and by causing the increased secretion of factors that can affect other cells such as cytokines, interleukins and growth factors.The first CAR-T cell therapies were FDA-approved in 2017,and there are now 5 approved CAR-T therapies.

References

Ordered List