Source 1primary paper2023MED
Toolkit/synNotch reporter system
synNotch reporter system
Also known as: synNotch reporter system, synthetic Notch (synNotch) system
Taxonomy: Mechanism Branch / Architecture. Workflows sit above the mechanism and technique branches rather than replacing them.
Summary
Here, using the synthetic Notch (synNotch) receptor that tethers antigen binding to customized transgene expression, we linked intratumoral immune-cancer cell communication to a simple secreted reporter blood test... Our synNotch reporter system allows for the monitoring of antigen-dependent intratumoral immune-cancer cell interactions through a simple and convenient blood test.
Usefulness & Problems
No literature-backed usefulness or problem-fit explainer has been materialized for this record yet.
Published Workflows
Objective: Engineer immune cells so that antigen-dependent intratumoral immune-cancer cell communication produces a blood-detectable secreted reporter signal.
Why it works: The workflow is based on using synNotch to convert antigen recognition on cancer cells into induced expression of a secreted reporter, allowing local immune-tumor contact to be read out through blood SEAP activity.
synNotch-mediated coupling of antigen binding to customized transgene expressionSEAP secretion after CD19-dependent receptor activationsequential lentiviral transductionco-culture reporter assaymouse in vivo blood reporter assay
Stages
- 1.Engineering of synNotch reporter Jurkat T cells(library_build)
This stage creates the engineered immune cells needed for downstream antigen-dependent reporter testing.
Selection: Generate cells carrying both the anti-CD19 synNotch receptor and the synNotch response element encoding SEAP.
- 2.In vitro antigen-specific co-culture screen(functional_characterization)
This stage tests whether the engineered circuit responds selectively to the intended antigen in a controlled co-culture setting before animal experiments.
Selection: Measure whether engineered cells produce elevated SEAP in response to CD19-positive but not CD19-negative Nalm6 cells.
- 3.In vivo blood reporter validation(in_vivo_validation)
This stage determines whether the antigen-dependent reporter circuit can be read out through blood in a tumor-bearing mouse model.
Selection: Test whether intratumoral delivery of engineered T cells yields blood-detectable SEAP selectively in mice bearing CD19-positive tumors.
Steps
- 1.Sequential lentiviral transduction of anti-CD19 synNotch receptorengineered receptor component
Introduce the antigen-sensing synNotch receptor into Jurkat T cells.
The receptor component is required as part of the two-component engineered circuit before reporter testing can occur.
- 2.Sequential lentiviral transduction of synNotch response element encoding SEAPreporter output component
Add the SEAP output module needed to convert synNotch activation into a measurable secreted signal.
The response element completes the two-component reporter circuit needed for downstream co-culture and in vivo assays.
- 3.Co-culture engineered cells with CD19-positive and CD19-negative Nalm6 targetsengineered system under test
Test whether reporter activation depends on target antigen status.
This provides a controlled specificity check before moving to in vivo testing.
- 4.Establish CD19-positive and CD19-negative Nalm6 tumors in NSG mice
Create in vivo tumor contexts differing by target antigen status for reporter validation.
Tumor-bearing mice are required before intratumoral delivery of engineered T cells and blood-based reporter measurement.
- 5.Intratumoral injection of engineered T cells and blood SEAP measurementengineered system under test
Determine whether intratumoral antigen-dependent cell interactions generate a blood-detectable reporter signal in vivo.
This is the in vivo confirmation step after establishing tumor-bearing mice and prior in vitro specificity evidence.
Taxonomy & Function
Primary hierarchy
Mechanism Branch
Architecture: A composed arrangement of multiple parts that instantiates one or more mechanisms.
Mechanisms
antigen-dependent receptor activationinduced secretion of a reporter proteinsynthetic receptor-coupled transcriptional activationTechniques
No technique tags yet.
Target processes
recombinationValidation
Cell-freeBacteriaMammalianMouseHumanTherapeuticIndep. Replication
Observations
successMammalian Cell Lineapplication demoJurkat T cells
co-culture reporter assay
Inferred from claim c3 during normalization. Engineered Jurkat T cells co-cultured with CD19-positive but not CD19-negative Nalm6 cells show elevated SEAP in media. Derived from claim c3. Quoted text: Co-culture of engineered cells with CD19+, but not CD19-, Nalm6 cells, resulted in significantly elevated SEAP in media.
Source:
successMouseapplication demomouseNSG
blood SEAP activity assay
Inferred from claim c4 during normalization. Intratumoral injection of engineered T cells into mice bearing CD19-positive tumors produces elevated blood SEAP activity, whereas CD19-negative tumors do not. Derived from claim c4. Quoted text: Intratumoral injection of engineered T cells (1x10^7) resulted in significantly elevated blood SEAP activity in mice bearing CD19+ tumors (n = 7), but not CD19- tumors (n = 5).
Source:
CD19-negative tumor cohort size5 mice(=)CD19-positive tumor cohort size7 mice(=)engineered T cell dose10000000 cells
Supporting Sources
Ranked Claims
Intratumoral injection of engineered T cells into mice bearing CD19-positive tumors produces elevated blood SEAP activity, whereas CD19-negative tumors do not.
Intratumoral injection of engineered T cells (1x10^7) resulted in significantly elevated blood SEAP activity in mice bearing CD19+ tumors (n = 7), but not CD19- tumors (n = 5).
CD19-negative tumor cohort size 5 miceCD19-positive tumor cohort size 7 miceengineered T cell dose 10000000 cells
A CD19-targeted synNotch receptor can drive SEAP production upon binding to CD19 on cancer cells in vivo.
we engineered immune cells with a CD19-targeted synNotch receptor and demonstrated that binding to CD19 on cancer cells in vivo resulted in the production of secreted embryonic alkaline phosphatase (SEAP)
The synNotch reporter system is proposed as a broadly applicable monitoring platform for different target antigens in cell-based immunotherapies.
Continued development of this system for different target antigens of interest should provide a broadly applicable platform for improved monitoring of many cell-based immunotherapies
Engineered Jurkat T cells co-cultured with CD19-positive but not CD19-negative Nalm6 cells show elevated SEAP in media.
Co-culture of engineered cells with CD19+, but not CD19-, Nalm6 cells, resulted in significantly elevated SEAP in media.
The synNotch reporter system links antigen-dependent intratumoral immune-cancer cell communication to a secreted reporter blood test.
we linked intratumoral immune-cancer cell communication to a simple secreted reporter blood test
Approval Evidence
1 source4 linked approval claimsfirst-pass slug synnotch-reporter-system
Here, using the synthetic Notch (synNotch) receptor that tethers antigen binding to customized transgene expression, we linked intratumoral immune-cancer cell communication to a simple secreted reporter blood test... Our synNotch reporter system allows for the monitoring of antigen-dependent intratumoral immune-cancer cell interactions through a simple and convenient blood test.
Source:
in vivo applicationsupports
Intratumoral injection of engineered T cells into mice bearing CD19-positive tumors produces elevated blood SEAP activity, whereas CD19-negative tumors do not.
Intratumoral injection of engineered T cells (1x10^7) resulted in significantly elevated blood SEAP activity in mice bearing CD19+ tumors (n = 7), but not CD19- tumors (n = 5).
Source:
platform scopesupports
The synNotch reporter system is proposed as a broadly applicable monitoring platform for different target antigens in cell-based immunotherapies.
Continued development of this system for different target antigens of interest should provide a broadly applicable platform for improved monitoring of many cell-based immunotherapies
Source:
specificitysupports
Engineered Jurkat T cells co-cultured with CD19-positive but not CD19-negative Nalm6 cells show elevated SEAP in media.
Co-culture of engineered cells with CD19+, but not CD19-, Nalm6 cells, resulted in significantly elevated SEAP in media.
Source:
tool functionsupports
The synNotch reporter system links antigen-dependent intratumoral immune-cancer cell communication to a secreted reporter blood test.
we linked intratumoral immune-cancer cell communication to a simple secreted reporter blood test
Source:
Comparisons
No literature-backed comparison notes have been materialized for this record yet.
Ranked Citations
- 1.