Part:BBa_K2549005

anti-CD19

Anti-CD19 (αCD19) is a CD19 scFv. Its heavy-chain variable region (αCD19a) and light-chain variable region (αCD19b) are fused using a glycine-rich peptide linker (3 repeats of GGGGS, or G4S for short). It was used as the extracellular domain of the SynNotch, thus accomplishing the contact-dependent signal input against CD19 (surCD19 was used in our project, Part:BBa_K2549001).

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI site found at 444
  

Biology

Clinical significance of CD19

As summarized on wikipedia page: 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.

CAR-T CD19

CD19-targeted chimeric antigen receptor T cell therapy: 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.

cancer.gov: CAR T cells and TCR T cells are engineered to produce special receptors on their surfaces. They are then expanded in the laboratory and returned to the patient. Image credit: National Cancer Institute.

CAR T cell therapy can cause several worrisome. One of the most frequent is cytokine release syndrome (CRS). Ironically, CRS is considered an “on-target” effect of CAR T cell therapy. Patients experiencing severe CRS all had particularly high levels of IL-6, a cytokine that is secreted by T cells and macrophages in response to inflammation. And, doctors have been using IL-6 blockade to manage IL-6 CRS. Another potential side effect of CAR T cell therapy is a mass die off of B cells, known as B-cell aplasia. CD19 is expressed on normal B cells, which are responsible for producing antibodies that kill pathogens. These normal B cells are also often killed by the infused CAR T cells. To compensate, many patients must receive immunoglobulin therapy, which provides them with the necessary antibodies to fight off infections.

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

Please refer the original article for more details. 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).