Difference between revisions of "Part:BBa K2549001"

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We demonstrate surCD19 works as designed by the following two data.
 
We demonstrate surCD19 works as designed by the following two data.
  
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[[File:surCD19.png|600px|thumb|'''On the left:''' 293FT cells expressing surCD19 were plated and fixed, but without permeabilization. Fluorescent antibodies against myc-tag (labeled with AF488 dye) were used to perform the immunostaining. Cells were imaged by a confocal microscope and the middle optical section is shown. Because there is no permeabilization, only surface presented protein, facing outside environment, could be stained by the antibody. Clearly, surCD19 has the correct topological orientation and the containing myc-tag was recognized by the antibody.'''On the right:''' Cells were not plated on the glass, but analyzed by FACS Jazz. The HA tag on surCD19 was labeled by a green fluorescent antibody, and the CD19 was labeled by a red fluorescent antibody. The cells were co-stained before the cell sorting. Please note that myc, HA and CD19 extracellular domain on surCD19 were all successfully demonstrated here.]]
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'''On the left:''' 293FT cells expressing surCD19 were plated and fixed, but without permeabilization. Fluorescent antibodies against myc-tag (labeled with AF488 dye) were used to perform the immunostaining. Cells were imaged by a confocal microscope and the middle optical section is shown. Because there is no permeabilization, only surface presented protein, facing outside environment, could be stained by the antibody. Clearly, surCD19 has the correct topological orientation and the containing myc-tag was recognized by the antibody.
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'''On the right:''' Cells were not plated on the glass, but analyzed by FACS Jazz. The HA tag on surCD19 was labeled by a green fluorescent antibody, and the CD19 was labeled by a red fluorescent antibody. The cells were co-stained before the cell sorting. Please note that myc, HA and CD19 extracellular domain on surCD19 were all successfully demonstrated here.
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Revision as of 07:18, 7 October 2018


suface-expressed CD19

Surface-expressed CD19 (surCD19) is built by joining CD8α signal peptide, CD19 extracellular domain and the transmembrane region of PDGFRβ (from N terminal to C terminal). Additional HA tag on its N terminal and a Myc tag on its C terminal to facilitate detection by antibodies[1]. CD8α peptide guides synthesized fusion protein to pass the translocon[2] into the endoplasmic reticulum[3], and the fusion protein will be later sugar modified in Golgi[4], presented on the plasma membrane and located to the outside of the cell. Transmembrane region of PDGFRβ embeds surCD19 on the membrane. It was used as the antigen for Part:BBa_K2549005.

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 588
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI.rc site found at 757
    Illegal SapI site found at 273
    Illegal SapI.rc site found at 91

Biology

Clinical significance of CD19

As summarized on wikipedia page[5]: B-lymphocyte antigen CD19, also known as CD19 molecule (Cluster of Differentiation 19), B-Lymphocyte Surface Antigen B4, T-Cell Surface Antigen Leu-12 and CVID3 is a transmembrane protein that in humans is encoded by the gene CD19. In humans, CD19 is expressed in all B lineage cells, except for plasma cells, and in follicular dendritic cells. CD19 plays two major roles for B cells: (1) it acts as an adaptor protein to recruit cytoplasmic signaling proteins to the membrane; (2) it works within the CD19/CD21 complex to decrease the threshold for B cell receptor signaling pathways. Due to its presence on all B cells, it is a biomarker for B lymphocyte development, lymphoma diagnosis and can be utilized as a target for leukemia immunotherapies.

CD19-targeted chimeric antigen receptor T-cell therapy[6]: Acute lymphoblastic leukemia (ALL) remains difficult to treat, with minimal improvement in more than 2 decades. Adoptive transfer of T cells engineered to express a chimeric antigen receptor (CAR) has emerged as a powerful targeted immunotherapy. Complete remission rates as high as 90% have been reported in children and adults with relapsed and refractory ALL treated with CAR-modified T cells targeting the B-cell–specific antigen CD19. For more details, please check Maude SL et al.

α-CD19 works extremely well in Royal KT et al 2016

Please refer the original article for more details[7]. Below is our summary of their article to explain why we focus on CD19.

Royal KT at al stated: SynNotch receptors have a custom ligand binding domain that detects a cell-surface antigen of interest (e.g., scFvs targeted to CD19 or Her2 or nanobodies to GFP), the core regulatory region of Notch that controls proteolysis, and a cytoplasmic orthogonal transcription factor (e.g., Gal4 VP64). The corresponding response elements for the orthogonal transcription factor controlling custom transcriptional programs are also engineered into the T cell.
Royal KT at al stated: CD4+ and CD8+ primary human T cells were engineered with the α-CD19 synNotch Gal4VP64 receptor and 5x Gal4 response elements control- ling the expression of a BFP reporter. Histogram showing selective induction of the BFP reporter in α-CD19 synNotch receptor receiver CD4+ T cells in response to stimulation with sender cells with CD19- or CD19+ K562s.
Royal KT at al stated: CD4+ AND CD8+ primary human T cells were engineered with the α-CD19 nanobody synNotch Gal4VP64 receptors and 5x Gal4 response elements controlling the expression of a BFP reporter. The percentages of synNotch T cells that upregulate the BFP reporter after 24 hr of stimulation with the indicated sender cells is given (n >= 3 for all conditions, error bars, SEM).
Royal KT at al stated: CD4+ T cells were engineered with the α-CD19 synNotch Gal4VP64 receptor and the corresponding response elements controlling the expression of either IL-2, IL-10, IL-12, or combined IL-2/MIP-1a. The cells were co-cultured with target CD19+ K562s or CD19- non-target K562s.
Royal KT at al stated: CD4+ α-CD19 synNotch T cells were engineered to regulate the expression Tbet and thus Th1 fate choice by T cells. The synNotch T cells were co-cultured with target CD19+ or non-target CD19- K562 cells for 11 days to determine if synNotch driven Tbet expression could skew CD4+ T cells to Th1 fate in a CD19- dependent manner.
Royal KT at al stated: (C) Histograms showing the selective expression of Tbet T2A EGFP after 24 hr of CD4+ α-CD19 synNotch T cells with CD19+ K562s (representative of at least three experiments). (D) Two-dimensional dot plots of intracellular stained CD4+ α-CD19 synNotch Gal4VP64 T cells for Tbet and IFNg after 11 days of culture with either CD19+ or CD19- K562s. T cells were stimulated with PMA/Ionomycin for 4 hr prior to staining to drive cytokine production (representative of at least three experiments). (E) The percentage of IFNg+ (Th1) T cells after 11 days of the indicated treatment (n >= 3 for all treatments, error bars, SEM, significance determined by Student’s t test, n.s. p >= 0.05).
Royal KT at al stated: CD4+ T cells were engineered with the α-CD19 synNotch receptor controlling the expression of PD-L1 and IL-10.
Royal KT at al stated: Quantification of the percentage of synNotch T cells that express PD-L1 and intracellular IL-10 after co-culture with CD19+ or CD19- K562s for 24 hr is given. The amount of IL-10 in the supernatant was also determined by ELISA (n = 3; error bars, SEM).
Royal KT at al stated: NSG mice were subcutaneously injected with CD19- non-target K562s and target CD19+ in the left and right flank, respectively. α-CD19 synNotch T cells in control of IL-2 iRES mCherry expression were injected into the mice after tumors were established and tumors were harvested at the indicated time point to determine whether the synNotch T cells had infiltrated the tumor and expression of IL-2 and mCherry reporter was induced.
Royal KT at al stated: Histograms of IL-2 IRES mCherry reporter levels in tumor and spleen infiltrated CD4+ synNotch T cells injected i.v. showing selective expression of the mCherry reporter in target CD19+ tumors (data representative of three replicate mice).
surCD19 works as we designed

Please note that surCD19 expressing cells are used as the target in our project. We were inspired by CD19+ K562 cells used in Royal KT et al, where they were over-expressing the intact CD19 molecule.

We used our previous strategy in surEGFP (Part:BBa_K2446051).

  • Using the same signal peptide as surEGFP but no the endogenous of CD19; to ensure comparable membrane targeting efficient as surEGFP
  • Endogenous transmembrane of CD19 is weak resulting high cytoplasmic signal, thus we replace it with the transmembrane domain from PDGF&beta.
  • Adding extra tags, as in Royal KT et al 2016, for immuno-staining and immuno-blocking to detect the expression level and its localization

We demonstrate surCD19 works as designed by the following two data.

On the left: 293FT cells expressing surCD19 were plated and fixed, but without permeabilization. Fluorescent antibodies against myc-tag (labeled with AF488 dye) were used to perform the immunostaining. Cells were imaged by a confocal microscope and the middle optical section is shown. Because there is no permeabilization, only surface presented protein, facing outside environment, could be stained by the antibody. Clearly, surCD19 has the correct topological orientation and the containing myc-tag was recognized by the antibody.On the right: Cells were not plated on the glass, but analyzed by FACS Jazz. The HA tag on surCD19 was labeled by a green fluorescent antibody, and the CD19 was labeled by a red fluorescent antibody. The cells were co-stained before the cell sorting. Please note that myc, HA and CD19 extracellular domain on surCD19 were all successfully demonstrated here.


References

  1. ↑ Engineering Customized Cell Sensing and Response Behaviors Using Synthetic Notch Receptors. Morsut L, Roybal KT, Xiong X, ..., Thomson M, Lim WA. Cell, 2016 Feb;164(4):780-91 PMID: 26830878; DOI: 10.1016/j.cell.2016.01.012
  2. ↑ https://en.wikipedia.org/wiki/Translocon
  3. ↑ https://en.wikipedia.org/wiki/Endoplasmic_reticulum
  4. ↑ https://en.wikipedia.org/wiki/Golgi_apparatus
  5. ↑ https://en.wikipedia.org/wiki/CD19
  6. ↑ CD19-targeted chimeric antigen receptor T-cell therapy for acute lymphoblastic leukemia. Maude SL, Teachey DT, Porter DL, Grupp SA. Blood, 2015 Jun;125(26):4017-23 PMID: 25999455; DOI: 10.1182/blood-2014-12-580068
  7. ↑ Engineering T Cells with Customized Therapeutic Response Programs Using Synthetic Notch Receptors. Roybal KT, Williams JZ, Morsut L, ..., McNally KA, Lim WA. Cell, 2016 Oct;167(2):419-432.e16 PMID: 27693353; DOI: 10.1016/j.cell.2016.09.011