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CD3×5T4 In Vitro Activation and Cytotoxicity QSP

Fixed-effect in vitro model linking CD3×5T4 immune-synapse formation to T-cell activation, differentiation, and delayed tumor-cell killing.

An In Vitro Quantitative Systems Pharmacology Platform for Characterizing CD3-Bispecific Antibody-Mediated T-Cell Activation and Tumor Cell Cytotoxicity ↗

The AAPS Journal

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MODEL IN MOTION 18 sec

How does the effector-to-target ratio change effector cells and tumor killing?

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MDA-MB-468 synapse-to-killing cascade, E:T 4:1

Population fixed-effect 72-hour in vitro culture at 6.86 nM DuoBody-CD3x5T4 and the default 4:1 effector-to-target ratio.

0–72 hour

Scenario details

MDA-MB-468 synapse-to-killing cascade, E:T 4:1

Population fixed-effect 72-hour in vitro culture at 6.86 nM DuoBody-CD3x5T4 and the default 4:1 effector-to-target ratio.

Readouts

Immune-synapse trimers per T cell · trimers/T cell
Read together with total effector-cell abundance: a high per-cell trimer signal does not by itself imply a large aggregate killing drive.
Activated or effector T cells · %
An in vitro state fraction, not a clinical response rate or a cytokine-release measurement.
Effector T cells · cells/well
This reflects the source differentiation mechanism and should not be interpreted as tumor infiltration in vivo.
Tumor-cell cytotoxicity · %
A model-derived in vitro cytotoxicity readout; it is not a clinical tumor-response endpoint.
Viable and apoptotic tumor cells · cells/well
Dead tumor cells are excluded after leaving the final apoptosis stage.

Study purpose

  • How does the reported 6.86 nM condition propagate from synapse formation to T-cell activation, effector accumulation, and delayed cytotoxicity over 72 hours?

Assumptions

  • Uses population fixed effects rather than individual-culture fits.
  • Concentrations describe the in vitro assay.

Reference data

  • Reference curves are digitized. Repeated renderings are not independent observations.

Inputs changed since the displayed result. Run to update the comparison.

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Binding

DuoBody-CD3x5T4 value; the high-affinity bsIgG1 variant is 16 nM. Values between the endpoints are exploratory and values outside them are outside this runtime contract.

nM

Suggested: 342–1,020. You can explore beyond this range.

683 → —
Estimated population effect

MDA-MB-468 cell-line group 3 value.

molecule/cell

Suggested: 1.07–3.2. You can explore beyond this range.

2.13 → —

Upper rate of trimer-dependent resting-to-activated T-cell conversion in the fixed-effect model.

1/hr

Suggested: 0.0053–0.0159. You can explore beyond this range.

0.0106 → —

Aggregate trimer load across effector T cells producing half-maximal per-effector killing drive.

molecule

Suggested: 3,550–1.06e+4. You can explore beyond this range.

7098 → —

MDA-MB-468 cell-line group 4 and no-donor/donor group 2 value.

1/(h·cell)

Suggested: 6.50e-6–1.95e-5. You can explore beyond this range.

0.000013 → —
Simulation time
h
h
72 → 168

Time between reported samples.

h

1 change · Variation 1

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Biology

A CD3×5T4 bispecific antibody bridges a resting T cell and a 5T4-positive tumor cell. Per-cell trimers activate and expand T cells, which differentiate into effectors. Effector cells push proliferating tumor cells through three apoptotic transit stages, producing activation and cytotoxicity readouts.

A CD3×5T4 bispecific antibody bridges a resting T cell and a 5T4-positive tumor cell. Per-cell trimers activate and expand T cells, which differentiate into effectors. Effector cells push proliferating tumor cells through three apoptotic transit stages, producing activation and cytotoxicity readouts.

Model details

33 parameters · 17 states · 17 equations · 18 derived outputs

Classification & API access
Therapeutic
DuoBody-CD3x5T4, bsIgG1-CD3x5T4
Modality
Bispecific antibody / T-cell engager
Target
CD3, 5T4
Disease / scope
In vitro solid-tumor cell-line assay (not disease-specific)
ParameterDefaultUnitDescription
Activated t cell doubling timeDT_Ta_hr12hrActivated t cell doubling time coefficient used by the released system of model equations.
Tumor cell doubling timeDT_tumor_hr40hrMDA-MB-468 default.
Trimer threshold for half-maximal activationEC50_activation_trimers_per_tcell2.13molecule/cellMDA-MB-468 cell-line group 3 value.
Generation count for half maximal differentiationEC50_differentiation_generations3—Generation count for half maximal differentiation at which the corresponding modeled effect reaches half of its maximum.
Aggregate trimer threshold for half-maximal killingEC50_killing_total_trimers7098moleculeAggregate trimer load across effector T cells producing half-maximal per-effector killing drive.
T cell activation hill coefficientGamma_activation0.623—T cell activation hill coefficient coefficient used by the released system of model equations.
Tumor killing hill coefficientGamma_killing1.63—Tumor killing hill coefficient governing loss, degradation, or removal in the modeled biological process.
Activated t cell half-lifeHL_Ta_hr48hrActivated t cell half-life coefficient used by the released system of model equations.
Effector t cell half-lifeHL_Te_hr48hrEffector t cell half-life coefficient used by the released system of model equations.
Resting t cell half-lifeHL_Tr_hr168hrResting t cell half-life coefficient used by the released system of model equations.
CD3-arm 2D dissociation constant — high-affinity endpointKD_CD3_2D_high_molecules_per_cell11088molecules/cellCD3-arm 2D dissociation constant — high-affinity endpoint describing equilibrium binding affinity in the modeled interaction.
CD3-arm 2D dissociation constant — low-affinity endpointKD_CD3_2D_low_molecules_per_cell174molecules/cellCD3-arm 2D dissociation constant — low-affinity endpoint describing equilibrium binding affinity in the modeled interaction.
Measured high CD3-arm 3D dissociation endpointKD_CD3_3D_high_nM683nMMeasured high CD3-arm 3D dissociation endpoint describing equilibrium binding affinity in the modeled interaction.
Measured low CD3-arm 3D dissociation endpointKD_CD3_3D_low_nM16nMMeasured low CD3-arm 3D dissociation endpoint describing equilibrium binding affinity in the modeled interaction.
CD3-arm 3D dissociation constantKD_CD3_3D_nM683nMDuoBody-CD3x5T4 value; the high-affinity bsIgG1 variant is 16 nM. Values between the endpoints are exploratory and values outside them are outside this runtime contract.
5T4-arm 2D dissociation constantKD_TAA_2D_molecules_per_cell27molecules/cell5T4-arm 2D dissociation constant describing equilibrium binding affinity in the modeled interaction.
5T4-arm 3D dissociation constantKD_TAA_3D_nM3nM5T4-arm 3D dissociation constant describing equilibrium binding affinity in the modeled interaction.
Maximum T-cell activation rateMax_activation_per_hr0.01061/hrUpper rate of trimer-dependent resting-to-activated T-cell conversion in the fixed-effect model.
Maximum t cell differentiation rateMax_differentiation_per_hr0.2681/hrMaximum t cell differentiation rate coefficient used by the released system of model equations.
Maximum per-effector tumor killing rateMax_kill_per_effector_hr0.0000131/(h·cell)MDA-MB-468 cell-line group 4 and no-donor/donor group 2 value.
Baseline CD3 abundancebase_CD3_molecules_per_tcell30000molecule/cellBaseline CD3 abundance coefficient used by the released system of model equations.
Baseline 5 T4 abundancebase_TAA_molecules_per_tumor31582molecule/cellMDA-MB-468 default from Supplement Table S1.
CD3 side complex internalization rateint_CD3_per_hr0.0661/hrCD3 side complex internalization rate coefficient used by the released system of model equations.
5 T4 side complex internalization rateint_TAA_per_hr0.0661/hr5 T4 side complex internalization rate coefficient used by the released system of model equations.
CD3 degradation ratekdeg_CD3_per_hr0.0661/hrCD3 degradation rate governing loss, degradation, or removal in the modeled biological process.
5 T4 degradation ratekdeg_TAA_per_hr0.0661/hr5 T4 degradation rate governing loss, degradation, or removal in the modeled biological process.
CD3 dissociation ratekoff_CD3_per_hr721/hrCD3 dissociation rate governing molecular dissociation in the binding model.
5 T4 dissociation ratekoff_TAA_per_hr3.7741/hr5 T4 dissociation rate governing molecular dissociation in the binding model.
Apoptosis transit ratektr_apoptosis_per_hr11/hrApoptosis transit rate coefficient used by the released system of model equations.
Molecule to medium concentration conversionmolecule_to_nM1.1e-11nM/moleculeMolecule to medium concentration conversion coefficient used by the released system of model equations.
Immune synapse areasynapse_area_um212.56um²Immune synapse area coefficient used by the released system of model equations.
T cell radiustcell_radius_um3.5umT cell radius coefficient used by the released system of model equations.
Tumor cell radiustumor_radius_um10umTumor cell radius coefficient used by the released system of model equations.

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