Part:BBa_K5017000
Chimeric Antigen Receptor (CAR) targeting Mesothelin (MSLN) coupled with GFP-BleoR
Our CAR construct is based on previous work of evaluation of different 2nd and 3rd generation CAR constructs that improve natural killer (NK) cell mediated killing by targeting mesothelin. Researchers identified a CAR containing the transmembrane domain of NKG2D, the 2B4 costimulatory domain, and the CD3z signaling domain to mediate the strongest antigen-specific NK cells signaling among all of the constructs. So we designed a sequence about 3 kb long to be cloned into a lentiviral vector. This sequence consists of:
--CD8a Leader domain: a critical component in the synthesis and membrane insertion of the CD8α protein. CD8α is a cell surface glycoprotein primarily found on the surface of cytotoxic T-cells, where it plays a crucial role in immune response regulation. It is responsible for guiding the nascent CD8α protein through the cellular secretory pathway and ensuring its proper localization to the cell membrane.
--Anti-mesothelin scFv: is a specialized antibody fragment engineered to target the mesothelin protein, which is often overexpressed in pancreatic cancer. The choice of the anti-mesothelin scFv is very critical for the CAR therapy efficiency. Taking into consideration this fact we chose to use a scFv sequence that binds to a juxtamembrane region of mesothelin at the site where it attaches to the cell membrane and is not inactivated by shed MSLN found at high concentrations inside tumors and in ascites and pleural fluid of cancer patients.
--CD8a Hinge domain: is a critical structural element within the CD8α protein. This hinge domain serves as a flexible linker between the CD8α protein's extracellular antigen-binding domain and its transmembrane region, allowing for the necessary conformational changes that occur during T-cell receptor (TCR) interactions with major histocompatibility complex (MHC) molecules on antigen-presenting cells. These dynamic structural adaptations are essential for the flexibility of the scFv domain of the CAR.
--NKG2D Transmembrane domain: is a critical structural component of the NKG2D receptor protein found on the surface of immune cells, particularly Natural Killer (NK) cells and some T-cells. This hydrophobic region spans the cell membrane, anchoring the NKG2D protein in the cell's lipid bilayer. The transmembrane domain plays a pivotal role in signal transduction and receptor function by facilitating the proper positioning and orientation of the NKG2D receptor on the cell surface. This ensures that NKG2D can interact with its ligands on target cells, initiating intracellular signaling pathways that lead to immune responses, including target cell lysis or cytokine release.
--2B4 domain: refers to the extracellular domain of the CD244 receptor. CD244 is a cell surface receptor primarily expressed on NK-cells, some T-cells, and other immune cells. The 2B4 domain plays a crucial role in immune cell interactions and regulation. It serves as a binding site for the CD48 ligand found on antigen-presenting cells, allowing for receptor-ligand interactions that can either activate or inhibit immune responses, depending on the context.
--CD3z domain: is a signaling subunit that contains immunoreceptor tyrosine-based activation motifs (ITAMs). When the CAR recognizes a specific antigen, CD3z becomes phosphorylated, initiating a cascade of intracellular signaling events that ultimately lead to NK-cell activation. This activation is a fundamental step in the immune response, as it triggers NK-cells to proliferate, release cytokines, and engage in various immune functions to combat infections or respond to abnormal cells, such as cancerous cells.
--IRES Element: which stands for Internal Ribosome Entry Site is a crucial genetic structure found within certain messenger RNA (mRNA) molecules. IRES elements are essential for the regulation of gene expression and protein synthesis, particularly in eukaryotic cells. Unlike the conventional cap-dependent translation initiation, where ribosomes start protein synthesis at the mRNA's 5' cap structure, IRES allows ribosomes to initiate translation internally, typically within the 5' untranslated region (UTR) of the mRNA. We used this element to express GFP from one polycistronic mRNA.
--GFP::Zeo: often referred to as Green Fluorescent Protein (GFP) fused with Zeocin resistance, is a powerful molecular biology tool used in various biotechnological and biomedical applications. GFP, originally derived from a bioluminescent jellyfish, is a naturally occurring fluorescent protein widely employed as a visual marker to track and study protein localization, gene expression, and cellular processes in living organisms. The fusion of GFP with Zeocin resistance confers resistance to the antibiotic Zeocin, allowing for the selection and maintenance of cells that express the GFP-tagged protein of interest. This dual-function fusion protein has become invaluable in molecular and cell biology research, as it enables the visualization of specific proteins within living cells while facilitating the stable selection of those cells in culture.
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal PstI site found at 451
- 12INCOMPATIBLE WITH RFC[12]Illegal PstI site found at 451
- 21COMPATIBLE WITH RFC[21]
- 23INCOMPATIBLE WITH RFC[23]Illegal PstI site found at 451
- 25INCOMPATIBLE WITH RFC[25]Illegal PstI site found at 451
Illegal NgoMIV site found at 2949
Illegal NgoMIV site found at 3010 - 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI.rc site found at 1012
| None |