Difference between revisions of "Part:BBa K2549001"

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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

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 could be used as the target. 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: xx

On the right: xx

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