Difference between revisions of "Team:SMMU-China/Demonstrate"

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             TTZ.CAR receptor (BBa_K3132022), respectively. Unpaired cysteine 164 within the CD8a hinge region was
 
             TTZ.CAR receptor (BBa_K3132022), respectively. Unpaired cysteine 164 within the CD8a hinge region was
 
             replaced with a serine to increase CAR expression as reported previously<sup>[1]</sup>.</p>
 
             replaced with a serine to increase CAR expression as reported previously<sup>[1]</sup>.</p>
         <img style="width: 40%;" src="https://static.igem.org/mediawiki/2019/4/44/T--SMMU-China--demonstrate_1.png"
+
         <img style="width: 60%;" src="https://static.igem.org/mediawiki/2019/4/44/T--SMMU-China--demonstrate_1.png"
 
             alt="smmu_demonstrate_1" style="margin: 10px 0px;">
 
             alt="smmu_demonstrate_1" style="margin: 10px 0px;">
 
         <center>Fig. 1 Design of CAR</center>
 
         <center>Fig. 1 Design of CAR</center>
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             by flow cytometry. The flow cytometry results suggested that lentiviral vectors effectively transduced NK-92
 
             by flow cytometry. The flow cytometry results suggested that lentiviral vectors effectively transduced NK-92
 
             cells (Fig. 2).</p>
 
             cells (Fig. 2).</p>
         <img src="https://static.igem.org/mediawiki/2019/f/f6/T--SMMU-China--demonstrate_2.png" alt="smmu_demonstrate_2"
+
         <img style="width: 60%;" src="https://static.igem.org/mediawiki/2019/f/f6/T--SMMU-China--demonstrate_2.png" alt="smmu_demonstrate_2"
 
             style="margin: 10px 0px;">
 
             style="margin: 10px 0px;">
 
         <center>Fig. 2 Expression of chimeric receptors on NK-92 cells</center>
 
         <center>Fig. 2 Expression of chimeric receptors on NK-92 cells</center>
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             10:1, respectively). EGFR and HER2-negative MCF-7 cells were not lysed by any of the NK-92 derivatives (Fig.
 
             10:1, respectively). EGFR and HER2-negative MCF-7 cells were not lysed by any of the NK-92 derivatives (Fig.
 
             3).</p>
 
             3).</p>
         <img src="https://static.igem.org/mediawiki/2019/5/58/T--SMMU-China--demonstrate_3.png" alt="smmu_demonstrate_3"
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         <img style="width: 60%;" src="https://static.igem.org/mediawiki/2019/5/58/T--SMMU-China--demonstrate_3.png" alt="smmu_demonstrate_3"
 
             style="margin: 10px 0px;">
 
             style="margin: 10px 0px;">
 
         <center>Fig. 3 Killing activity of CAR-NK cells in response to tumour cells.</center>
 
         <center>Fig. 3 Killing activity of CAR-NK cells in response to tumour cells.</center>
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             gene circuits. The double positive cells were termed as MTZ-synNotch.IC9.UAS-CTX.I2his cells. Similar
 
             gene circuits. The double positive cells were termed as MTZ-synNotch.IC9.UAS-CTX.I2his cells. Similar
 
             methods were used to sort the TTZ-synNotch.IC9.UAS-CTX.I2FLAG cells. </p>
 
             methods were used to sort the TTZ-synNotch.IC9.UAS-CTX.I2FLAG cells. </p>
         <img src="https://static.igem.org/mediawiki/2019/1/11/T--SMMU-China--demonstrate_4.png" alt="smmu_demonstrate_4"
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         <img style="width: 60%;" src="https://static.igem.org/mediawiki/2019/1/11/T--SMMU-China--demonstrate_4.png" alt="smmu_demonstrate_4"
 
             style="margin: 10px 0px;">
 
             style="margin: 10px 0px;">
 
         <center>Fig. 4 Construction of synNotch-NK cells. a, Design of EGFR-tareting synNotch-NK cell. b, Design of
 
         <center>Fig. 4 Construction of synNotch-NK cells. a, Design of EGFR-tareting synNotch-NK cell. b, Design of
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             is an indicator of the medical conditions and is a signal molecule to trigger the response of external
 
             is an indicator of the medical conditions and is a signal molecule to trigger the response of external
 
             device cells(Figure 5b).</p>
 
             device cells(Figure 5b).</p>
         <img src="https://static.igem.org/mediawiki/2019/c/c3/T--SMMU-China--demonstrate_5.png" alt="smmu_demonstrate_5"
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         <img style="width: 60%;" src="https://static.igem.org/mediawiki/2019/c/c3/T--SMMU-China--demonstrate_5.png" alt="smmu_demonstrate_5"
 
             style="margin: 10px 0px;">
 
             style="margin: 10px 0px;">
 
         <center>Fig. 5 Characterization of core device cells. a, Killing activity of synNotch-NK cells was compared with
 
         <center>Fig. 5 Characterization of core device cells. a, Killing activity of synNotch-NK cells was compared with

Revision as of 12:16, 21 October 2019

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Demonstrate
Proof and evidence.

Establishment of EGFR and HER2 Cancer Cell Models

To demonstrate the feasibility of our synthetic device, we first established cell models that were able to stably express different antigens on their surfaces. We chose epidermal growth factor receptor (EGFR) and human epidermal growth factor receptor 2 (HER2) as representative antigens for they are frequently used as drug targets and biomarkers in cancers. MCF-7 cells were transfected with the gene of EGFR or HER2 so that MCF-7 EGFR cells and MCF-7 HER2 cells were yielded and used for further study. The EGFR and HER2 expression levels of these cells were measured by flow cytometry, and the results are shown in Table 1.



Table 1. EGFR and HER2 expression on cell lines

Cancer cell line Isotype control EGFR HER2
MCF-7 2.6 2.4 2.5
MCF-7 EGFR 16.6 395.5 24.6
MCF-7 HER2 22.3 27.5 358.5

Design and Characterization of Chimeric Antigen Receptor (CAR)

The Design of CAR

The second generation CAR design, which fused a scFv, a CD8a hinge, a CD8 transmembrane domain, a 4-1BB intracellular domain and a CD3ζ chain in tandem was used in our project (Fig. 1). We designed two chimeric receptors that contain scFv derived from antibodies that recognize human EGFR and HER2. Cetuximab (anti-EGFR) and Trastuzumab (anti-HER2) were chosen because these antibodies have been found to be safe in patients when administered as targeted drugs. And these two receptors were named CTX.CAR (BBa_K3132021) and TTZ.CAR receptor (BBa_K3132022), respectively. Unpaired cysteine 164 within the CD8a hinge region was replaced with a serine to increase CAR expression as reported previously[1].

smmu_demonstrate_1
Fig. 1 Design of CAR

Deliver CAR into NK-92 cells

We used the lentiviral vector for transfecting NK-92 following clinically validated techniques[2]. The DNA sequences of the various fusion constructs were cloned into the pHR vector backbone under the control of a PGK promoter. The receptors were labelled extracellularly with a Myc epitope to allow detection by flow cytometry. The flow cytometry results suggested that lentiviral vectors effectively transduced NK-92 cells (Fig. 2).

smmu_demonstrate_2
Fig. 2 Expression of chimeric receptors on NK-92 cells

Antigen-specific Cell Killing Activity of CAR NK-92 Cells

Next, we investigated the anti-tumour potential of the transduced NK-92 cells by standard 51Cr-release assays. CAR NK-92 cells were cocultured with MCF-7 cells (EGFR and HER2-negative cells), MCF-7 EGFR cells (a derivative engineered to express EGFR), and MCF-7 HER2 cells (a derivative engineered to express HER2) established previously. NK-92 cells transduced with cetuximab scFv (termed CTX.CAR-NK92) efficiently lysed EGFR-positive cells (18.9%, 50.8%, and 80.4% specific lysis at an E/T ratio of 1:1, 5:1 and 10:1, respectively). On the other hand, NK-92 cells transduced with trastuzumab scFv (termed TTZ.CAR-NK92) efficiently lysed HER2-positive cells(26.1%, 46.6%, and 81.2% specific lysis at an E/T ratio of 1:1, 5:1 and 10:1, respectively). EGFR and HER2-negative MCF-7 cells were not lysed by any of the NK-92 derivatives (Fig. 3).

smmu_demonstrate_3
Fig. 3 Killing activity of CAR-NK cells in response to tumour cells.

Design and Construction of Core Synthetic Device Based on Immune Cells

After verifying the killing activity of CAR NK cells, we next set out to construct the core cells of our Theranostic nanobots. Instead of directly using CARs to recognize antigens, we let the NK-92 cells first express synNotch receptors to identify antigens, then leading to the expression of CARs and other genes. It is because the reporters for the activation of CAR is not stable.

We constructed two lentiviral vectors for co-transfection of either anti-EGFR or anti-HER2 core cells (Fig. 4a and Fig. 4b). As for anti-EGFR core cell, the first vector encodes an EGFR-specific synNotch receptor bearing a Gal4-VP64 intracellular transcription activation domain (Fig. 4a). A scFv derived from the anti-EGFR antibody matuzumab was used in the synNotch receptor as it has a distinct epitope compared with that of cetuximab. The second vector we generated contained a fusion gene encoding the same EGFR-CAR and a His-tagged IL2 gene, under the control of the upstream activating sequence (UAS) promoter that is activated by Gal4-VP64 released after engagement of the synNotch receptor. The two coding sequences were linked together using 2A sequence peptides6. Constitutively expressed blue fluorescent protein (BFP) was placed downstream of the inducible CAR transgene to identify transduced NK-92 cells (Fig. 4a).

Similar design was adopted for the HER2-antigen, we used an anti-HER2-domianⅠ antibody H2-18 for the synNotch receptor, which has a far distinct epitope compared with that of trastuzumab for TTZ.CAR. In the second vector, a FLAG tagged IL-2 is utilized to report the role of the cell (Fig. 4b).

NK-92 cells were co-transduced with both lentiviral vectors and co-transduction was verified by EGFR-Fc protein and BFP expression (Fig. 4c). Double positive NK-92 cells were enriched by fluorescence-activated cell sorting, and only about 10% of NK-92 cells were αEGFR+BFP+ and carried the full gene circuits. The double positive cells were termed as MTZ-synNotch.IC9.UAS-CTX.I2his cells. Similar methods were used to sort the TTZ-synNotch.IC9.UAS-CTX.I2FLAG cells.

smmu_demonstrate_4
Fig. 4 Construction of synNotch-NK cells. a, Design of EGFR-tareting synNotch-NK cell. b, Design of HER2-targeting synNotch-NK cell. c, Fluorescence-activated cell sorting of double-postitive cells.

Killing activity and Signal Molecules Secretion of Core Synthetic Devices

We next cultured sorted synNotch NK cells, and CAR-NK cells with MCF-7 EGFR, MCF-7 HER2 to test recognition of different antigen expressing tumor cells(Fig. 5a). Both CTZ.CAR-NK92 cells and TTZ.CAR-NK92 cells effectively killed EGFR+ or HER2+ cancer cells in 4 and 24 h co-cultures. By contrast, synNotch NK-92 cells recognized cancer cells expressing EGFR or HER2, and comparable lytic activity to CAR-NK-92 cells required 24 h of co-culture, consistent with previous reports showing that 12–24 h is required to fully upregulate CAR expression after engagement of the synNotch receptor[3,4].

In addition, the presence of antigen was sufficient to induce secretion of the IL-2-Tag protein at 24h, which is an indicator of the medical conditions and is a signal molecule to trigger the response of external device cells(Figure 5b).

smmu_demonstrate_5
Fig. 5 Characterization of core device cells. a, Killing activity of synNotch-NK cells was compared with CAR-NK cells. b, Core divice releases synthetic biomarkers.

References

1. Schönfeld, K. et al. Selective Inhibition of Tumor Growth by Clonal NK Cells Expressing an ErbB2/HER2-Specific Chimeric Antigen Receptor. Molecular Therapy the Journal of the American Society of Gene Therapy 23, 330 (2015).
2. Levine, B. L. et al. Gene transfer in humans using a conditionally replicating lentiviral vector. Proc Natl Acad Sci U S A 103, 17372-17377 (2006).
3. Roybal, K. et al. Engineering T Cells with Customized Therapeutic Response Programs Using Synthetic Notch Receptors. Cell 167, 419-432.e416 (2016).
4. Srivastava, S. et al. Logic-Gated ROR1 Chimeric Antigen Receptor Expression Rescues T Cell-Mediated Toxicity to Normal Tissues and Enables Selective Tumor Targeting. Cancer cell 35, 489-503 e488, doi:10.1016/j.ccell.2019.02.003 (2019).