Composite

Part:BBa_K5062007

Designed by: Christian Nelson Tejedor Vicera   Group: iGEM24_HKU-HongKong   (2024-09-27)


5th Generation Nanobody Chimeric Antigen Receptor (CAR) targeting GPC-3

Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal EcoRI site found at 2947
    Illegal SpeI site found at 1264
    Illegal PstI site found at 423
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal EcoRI site found at 2947
    Illegal SpeI site found at 1264
    Illegal PstI site found at 423
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal EcoRI site found at 2947
    Illegal BamHI site found at 54
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal EcoRI site found at 2947
    Illegal SpeI site found at 1264
    Illegal PstI site found at 423
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal EcoRI site found at 2947
    Illegal SpeI site found at 1264
    Illegal PstI site found at 423
    Illegal AgeI site found at 60
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 1019
    Illegal BsaI.rc site found at 524
    Illegal BsaI.rc site found at 2231
    Illegal SapI.rc site found at 2436

Overview

This is a Chimeric Anigen Receptor targeting GPC3 built for human macropages. It uses a nanobody instead of a traditional scFv, costimulatory domains to enhance phagocytosis, a control switch modulated by trimethoprim (TMP) and IFNγ as an polarisation factor keeping macrophages in an anti-tumor M1 state while guiding tumor-associated macropages from M2 towards M1 as well.


Usage and Biology

This sequence encodes an entire CAR polyprotein used for testing. Due to the complexity of this protein, it is unable to be cloned via the iGEM cloning standards. Rather, we recommend designing primers to clone this on to subsequent systems via NEBuilder as done by HKU-HongKong. Further, by using primers the RFP mCherry XL can also be removed allowing for removal if desired for use in downstream clinical applications.

This 5th Generation Chimeric Antigen Receptor has the following features:

  • An anti-GPC3 nanobody targeting domain enabling targeting of tumors such as hepatocellular carcinoma, squamous non-small cell lung cancer and breast cancer. Nanobodies hold advantages over traditional scFvs by being smaller, easier to engineer, and less immunogenic (Jin et al., 2023).
  • Optimized human co-stimulatory domains with CD19-FcγR Tandem and MegF10 inspired from the work of Morrissey, M. A. et al. (2018) and their work with mouse phagocytes. These co-stimulatory domains enhance phagocytosis, trogocytosis, engulfment of large targets, antigen presentation and macrophage micration.
  • Human IFNγ Armouring Domain allowing for the sustained M1 poloarization of macrophages enforcing their anti-tumor effect (Jorgovanovic et al., 2020). The co-expression of IFNγ also allows for the reprogramming of nearby tumor-associated macropahges (TAMs) within the tumor microenvironment (TME).
  • Variable control switch modulated by trimethoprim (TMP). A highlight of the 5th Generation CAR is the ability to control the strength of CAR expression by leveraging TMP-stabilized DD-L7Ae to trigger a strong conformational in 2x kink turn sequences on RNA, we are able to vary the concentration of TMP to effectively vary the strength of expression


A schematic demonstrating the architechture of earlier generations of Chimeric Antigen Receptor (Wu et al., 2020).


Components and Architecture

  • 2x Kink Turn - BBa_K4696005
  • Kozak Sequence - BBa_K4344028
  • CD8 Signal Peptide - BBa_K2549044
  • CD8 Hinge Domain - BBa_K5017003
  • CD28 Transmembrane Domain - BBa_K4803006
  • Human CD19-FcγR Tandem - BBa_K5062006
  • Human MegF10 - BBa_K5062005
  • P2A self-cleaving sequence - BBa_K1442039
  • Human IFNγ Armouring Domain - BBa_K4696020
  • DD-L7Ae - BBa_K5062002
  • mCherry XL RFP - BBa_K4241792


Results

In vitro characterisation of this part is currently ongoing prior to the wiki freeze. Full part characterisation is expected by the 2024 iGEM Giant Jambouree.

The study by Morrissey, M. A. et al. (2018) depics the improvement in phagocytotic ability with co-stimulatory domains such as FcγR and MegF10

The study by Cafferty, S. M. et al. (2020) depics the increase in expression as TMP decreases demonstrating the shut-off effect of this system.


References

Cafferty, S. M., De Temmerman, J., Kitada, T., Becraft, J. R., Weiss, R., Irvine, D. J., Devreese, M., De Baere, S., Combes, F., & Sanders, N. N. (2021). In vivo validation of a reversible small Molecule-Based switch for synthetic Self-Amplifying mRNA regulation. Molecular Therapy, 29(3), 1164–1173. https://doi.org/10.1016/j.ymthe.2020.11.010

Jin, B., Odongo, S., Radwanska, M., & Magez, S. (2023). Nanobodies: A Review of generation, Diagnostics and Therapeutics. International Journal of Molecular Sciences, 24(6), 5994. https://doi.org/10.3390/ijms24065994

Jorgovanovic, D., Song, M., Wang, L., & Zhang, Y. (2020). Roles of IFN-γ in tumor progression and regression: a review. Biomarker Research, 8(1). https://doi.org/10.1186/s40364-020-00228-x

Morrissey, M. A., Williamson, A. P., Steinbach, A. M., Roberts, E. W., Kern, N., Headley, M. B., & Vale, R. D. (2018). Chimeric antigen receptors that trigger phagocytosis. eLife, 7. https://doi.org/10.7554/elife.36688

Wagner, T. E., Becraft, J. R., Bodner, K., Teague, B., Zhang, X., Woo, A., Porter, E., Alburquerque, B., Dobosh, B., Andries, O., Sanders, N. N., Beal, J., Densmore, D., Kitada, T., & Weiss, R. (2018). Small-molecule-based regulation of RNA-delivered circuits in mammalian cells. Nature Chemical Biology, 14(11), 1043–1050. https://doi.org/10.1038/s41589-018-0146-9

Wu, L., Wei, Q., Brzostek, J., & Gascoigne, N. R. J. (2020). Signaling from T cell receptors (TCRs) and chimeric antigen receptors (CARs) on T cells. Cellular and Molecular Immunology, 17(6), 600–612. https://doi.org/10.1038/s41423-020-0470-3


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