Part:BBa_K4421003
scfv library with 10 individual colonies
This part is a phage display scfv library which consists of ten kinds of individual colonies. The sequence encoding the scFv antibodies generated from cetuximab, trastuzumab, CH65, 9.8B, 2F5, 7D11, 8D6, omalizumab, TE33, and R10 were chemically synthesized. The sequence information can be found in the Protein Data Bank. And we next aim to construct a CAR-NK-92 cell library of 10 kinds of unique CAR constructs with this part.
Usage and Biology
Antibody libraries can be constructed based on different sources, such as immunized animals or naturally immunized or infected humans, or naive immune systems, which can be derived from nonimmune natural or computational and synthetic sources. A single-chain variable fragment (scFv) is actually not 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 receptors). In our vitro experiments, the MCF-7 cells and the derivative cells were characterized, which have been used in our previous study. A small library of 10 CAR constructs comprising antigen-specific scFv(BBa K4421003) was constructed, termed as the NK-92CTX-TTZ library. And the sequence encoding the scFv antibodies was generated from cetuximab(CTX), trastuzumab(TTZ), CH65, 9.8B, 2F5, 7D11, 8D6, omalizumab, TE33, and R10.
Background and detail description
Tumor heterogeneity
Cancer is a dynamic disease. During the course of disease, cancers generally become more heterogeneous(Vendramin et al., 2021). As a result of this heterogeneity, the bulk tumour might include a diverse collection of cells harbouring distinct molecular signatures with differential levels of sensitivity to treatment. This heterogeneity might result in a non-uniform distribution of genetically distinct tumour-cell subpopulations across and within disease sites (spatial heterogeneity) or temporal variations in the molecular makeup of cancer cells (temporal heterogeneity)(Dagogo-Jack & Shaw, 2018). Heterogeneity provides the fuel for resistance; therefore, an accurate assessment of tumour heterogeneity is essential for the development of effective therapies. Traditional CAR-based immunotherapies tend to target only one or several targets at the same time, but none of them often lead to a lasting immune response due to tumor heterogeneity(Theoharis, 2021). The reason why we construct this part is to aim at solving tumor heterogeneity. At first, we intend to construct CAR-NK cells which carry 10 kinds of CAR and every CAR has a unique scfv sequence(BBa K4421003).
Antigen recognition domain
The antigen recognition domain is exposed to the outside of the cell, in the ectodomain portion of the receptor. It interacts with potential target molecules and is responsible for targeting the CAR-T cell to any cell expressing a matching molecule.
The antigen recognition domain is typically derived from the variable regions of a monoclonal antibody linked together as a single-chain variable fragment (scFv). An scFv is a chimeric protein made up of the light (VL) and heavy (VH) chains of immunoglobins, connected with a short linker peptide. These VL and VH regions are selected in advance for their binding ability to the target antigen (such as CD19). The linker between the two chains consists of hydrophilic residues with stretches of glycine and serine in them for flexibility as well as stretches of glutamate and lysine for added solubility. Single domain antibodies (e.g. VH, VHH) have been engineered and developed as antigen recognition domains in the CAR format due to their high transduction efficiency in T cells. In addition to antibody fragments, non‐antibody‐based approaches have also been used to direct CAR specificity, usually taking advantage of ligand/receptor pairs that normally bind to each other. Cytokines, innate immune receptors, TNF receptors, growth factors, and structural proteins have all been successfully used as CAR antigen recognition domains.
Sequence and Features
This part is a phage display scfv library which consists of ten kinds of individual colonies.For this reason,we can't put ten different sequences on this page.Instead,we create ten different basic part to record their sequence.They are BBa_K4421011, BBa_K4421012, BBa_K4421013, BBa_K4421014, BBa_K4421015, BBa_K4421016, BBa_K4421017, BBa_K4421018, BBa_K4421019 and BBa_K4421020.
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
References
Vendramin, R., Litchfield, K., & Swanton, C. (2021). Cancer evolution: Darwin and beyond. The EMBO Journal, 40(18), e108389.https://doi.org/10.15252/embj.2021108389
Dagogo-Jack, I., & Shaw, A. T. (2018). Tumour heterogeneity and resistance to cancer therapies. Nature Reviews. Clinical Oncology, 15(2), 81–94.https://doi.org/10.1038/nrclinonc.2017.166
Theoharis, S. (2021). Current State of the Art of Allogeneic CAR Approaches—Pile ’Em High and Sell ’Em Cheap. Journal of Pharmaceutical Sciences, 110(5), 1909–1914.https://doi.org/10.1016/j.xphs.2021.02.006
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