Antibody Information

General Information of This Antibody
Antibody ID
ANI0AXSHC
Antibody Name
Anit-TF mAb 25A3
Antibody Type
Monoclonal antibody (mAb)
Antibody Subtype
Chimeric IgG1-kappa
Antigen Name
Tissue factor (F3)
  Antigen Info 
The Activity Data of This Antibody
Antibody Activity Information 1 [1]
Internalization Rate (IR)
34
%
A431 cells CVCL_0037 
Antigen Expression Positive Tissue factor expression (TF+++/++; 380,000 TF receptor copy number)
Antibody Function Fluorescently labeled TF-specific antibody(25A3) to corroboate the cellular uptake, and the quantitative assay based on internalized fluorescence and quenched surface fluorescence.
Antibody Antigen Binding Assay A431 cells were seeded onto 8-well poly-D-lysine treated slides. One day later, the culture medium was replaced with medium containing ADC at 5 nM. After twenty hours of ADC exposure, the cells were fixed for 15 min at room temperature with 4 % paraformaldehyde. After three washes with PBS, the cells were permeabilized for 1 h with PBS containing 0.3 % Triton X-100 and 5 % normal goat serum. Next, the microtubule networks were stained for 3 h with anti-tubulin (11H10) rabbit mAb (Alexa Fluor 488 conjugate) in PBS containing 1 % BSA and 0.3 % Triton X-100. Internalization of anti-TF antibodies conjugated to A488. Upon labeling of MDA-MB-231 cells with the anti-TF antibody conjugates, the cells were pulsed at 37 C for up to 4 h prior to flow cytometry analysis. Percent internalization over time was calculated.
Antibody Activity Information 2 [1]
Half Maximal Inhibitory Concentration (IC50)
0.09
nM
A431 cells CVCL_0037 
Antigen Expression Positive Tissue factor expression (TF+++/++; 380,000 TF receptor copy number)
Antibody Function TF-specific antibody(25A3) complexed with a Fab fragment conjugated to monomethyl auristatin F (Fab:MMAF) can mediate internalization and toxin delivery into cells and trigger cell death in vitro.
Antibody Antigen Binding Assay Antibodies and an anti-human Fc Fab conjugated to the tubulin inhibitor mono-methyl auristatin F (MMAF, Moradec) were serially diluted, starting at 5 and 30 nmol/L, respectively. The anti-human Fc Fab conjugated to MMAF consisted of a polyclonal antibody specific to the Fc region of human IgGs with a DAR of 1.2 to 1.5. Plates were incubated for 3 to 5 days, followed by lysis in CellTiter-Glo (CTG) assay reagent. Cell viability of TF-positive A431 cell cultures three days after addition of a titration of antiTF antibody complexed with a Fab fragment against the human Fc conjugated to mono-methyl auristatin F (Fab:MMAF).
Antibody Activity Information 3 [1]
Half Maximal Inhibitory Concentration (IC50)
2.3
nM
MDA-MB-231 cells CVCL_0062 
Antigen Expression Positive Tissue factor expression (TF+++/++; 320,000 TF receptor copy number)
Antibody Function Evaluate the impact of TF-specific antibody (25A3) on TF signaing in cell cultures treated with FVIIa.
Antibody Antigen Binding Assay TF positive MDA-MB-231 cells that underwent a 2 h serum starvation with Leibovitzs L-15 medium were incubated with an 8-point 1:2.5 titration starting at 100 nM of anti-TF antibody. After 30 min at 37 C, FVIIa was added to the cells at a final concentration of 20 nM. Cell culture supernatants were harvested 5 h later and analyzed by ELISA for IL-8 as recommended. FVIIa-dependent TF-signaling in MDA-MB-231 cells pretreated with an anti-TF antibody titration was evaluated by measuring the concentration of IL8 in cell culture supernatants.
Antibody Activity Information 4 [1]
Half Maximal Inhibitory Concentration (IC50)
2.3
nM
MDA-MB-231 cells CVCL_0062 
Antigen Expression Positive Tissue factor expression (TF+++/++; 320,000 TF receptor copy number)
Antibody Function Evaluate the impact of TF-specific antibody (25A3) on TF signaing in cell cultures treated with FVIIa.
Antibody Antigen Binding Assay TF positive MDA-MB-231 cells that underwent a 2 h serum starvation with Leibovitzs L-15 medium were incubated with an 8-point 1:2.5 titration starting at 100 nM of anti-TF antibody. After 30 min at 37 C, FVIIa was added to the cells at a final concentration of 20 nM. Cell culture supernatants were harvested 5 h later and analyzed by ELISA for GM CSF as recommended. FVIIa-dependent TF-signaling in MDA-MB-231 cells pretreated with an anti-TF antibody titration was evaluated by measuring the concentration of GM-CSF in cell culture supernatants.
Antibody Activity Information 5 [1]
Half Maximal Effective Concentration (EC50)
0.08
nM
HCT 116 cells CVCL_0291 
Antigen Expression Positive Tissue factor expression (TF+++/++; 22,000 copies of surface)
Antibody Function Anti-TF antibody (25A3) bound to HCT-116 human colon cancer cell.
Antibody Antigen Binding Assay For cell-based antibody binding, approximately 1.2x105 HCT-116 cells were incubated with a twelve-point 1:3 dilution titration of anti-human TF IgG1 antibody starting at 250 or 100 nM for 2 h on ice. After 2 washes, cells labeled with anti-TF antibody were incubated for 30 min on ice with 150 nM of Phycoerythrin (PE) F(ab)2 fragment goat anti-human IgG, Fc-gamma fragment specific. After 2 washes, dead cells were labeled with TO-PRO-3 Iodide and samples were analyzed on a CytoFLEX flow cytometer or Novocyte flow cytometer.
Antibody Activity Information 6 [1]
Half Maximal Effective Concentration (EC50)
0.16
nM
HCT 116 cells CVCL_0291 
Antigen Expression Positive Tissue factor expression (TF+++/++; 22,000 copies of surface)
Antibody Function The cell-binding properties of the TF-specific antibody (25A3) moiety of the ADC and naked antibody.
Antibody Antigen Binding Assay Binding of antiTissue Factor (TF) antibodies and ADCs to human TF positive HCT-116 cells. For each anti-TF antibody or ADC, MFI of antibody or ADC bound to cells is plotted against antibody or ADC concentration. Reportable cell EC50s and their 95% confidence intervals are listed.
Antibody Activity Information 7 [1]
Half Maximal Effective Concentration (EC50)
0.17
nM
MDA-MB-231 cells CVCL_0062 
Antigen Expression Positive Tissue factor expression (TF+++/++; 320,000 TF receptor copy number)
Antibody Function Binding of anti-TF antibody (25A3) conjugated to Alexa Fluor 488 (highly stable fluorescent antibody) to MDA-MB-231 human breast cancer cells.
Antibody Antigen Binding Assay For cell-based antibody binding, approximately 1.2x105 MDA-MB-231 cells were incubated with a twelve-point 1:3 dilution titration of anti-human TF IgG1 antibody starting at 250 or 100 nM for 2 h on ice. After 2 washes, cells labeled with anti-TF antibody were incubated for 30 min on ice with 150 nM of Phycoerythrin (PE) F(ab)2 fragment goat anti-human IgG, Fc-gamma fragment specific. After 2 washes, dead cells were labeled with TO-PRO-3 Iodide and samples were analyzed on a CytoFLEX flow cytometer or Novocyte flow cytometer.
Antibody Activity Information 8 [1]
Half Maximal Effective Concentration (EC50)
0.22
nM
MDA-MB-231 cells CVCL_0062 
Antigen Expression Positive Tissue factor expression (TF+++/++; 320,000 TF receptor copy number)
Antibody Function TF-specific antibody (25A3) and ADC potently induced killing of A431 cells by ADCC.
Antibody Antigen Binding Assay Antibody-dependent cellular cytotoxicity (ADCC) A431 cells were plated on a microtiter plate. The following day, the cells were incubated with a ten-point 1:3 dilution titration of anti-TF antibodies or the ADCs starting at 50 nM. An ADCC effector-to-target cell ratio of 8:1 was added to each well and incubated for 6 h at 37 C. Luciferase Assay Reagent was added to each well to measure luminescence on an Envision plate reader. Antibody-dependent cellular cytotoxicity (ADCC) reporter luminescence was evaluated after a 6-hour incubation of the reporter Jurkat cell line with TF-positive A431 cells and a titration of anti-TF antibody or ADC. The ADCC reporter luminescence EC50 values for each anti-TF antibody or ADC are listed.
Antibody Activity Information 9 [1]
Half Maximal Effective Concentration (EC50)
0.5
nM
A431 cells CVCL_0037 
Antigen Expression Positive Tissue factor expression (TF+++/++; 380,000 TF receptor copy number)
Antibody Function Anti-TF antibody (25A3) bound to A431 epidermoid adenocarcinoma cell human colon cancer cell.
Antibody Antigen Binding Assay For cell-based antibody binding, approximately 1.2x105 A431 cells were incubated with a twelve-point 1:3 dilution titration of anti-human TF IgG1 antibody starting at 250 or 100 nM for 2 h on ice. After 2 washes, cells labeled with anti-TF antibody were incubated for 30 min on ice with 150 nM of Phycoerythrin (PE) F(ab)2 fragment goat anti-human IgG, Fc-gamma fragment specific. After 2 washes, dead cells were labeled with TO-PRO-3 Iodide and samples were analyzed on a CytoFLEX flow cytometer or Novocyte flow cytometer.
Antibody Activity Information 10 [1]
Half Maximal Effective Concentration (EC50)
1.1
nM
Antibody Function Kinetic measurements for the anti-TF antibodies,antibody affinity of 25A3.
Antibody Antigen Binding Assay ForteBio (Octet QK384, Pall ForteBio) affinity measurements.ForteBio affinity measurements were performed by loading IgGs on-line onto anti-hIgG capture (AHC) sensors. Sensors were equilibrated off-line in assay buffer for 30 min and then monitored on-line for 60 s for baseline establishment. Sensors with loaded IgGs were exposed to 100 nM recombinant TF (human or cynomolgus) for 3 min, then transferred to assay buffer for 3 min for off-rate measurement. Alternatively, binding measurements were obtained by loading biotinylated TF monomer onto streptavidin sensors followed by exposure to 100 nM antibody Fab in solution. Kinetic data was analyzed and fitted using a 1:1 Langmuir binding model and the KD was calculated by dividing the koff by the kon. ForteBio off-rates < 2x10-4 were reported as 2x10-4.
Antibody Activity Information 11 [1]
Dissociation Constant (Kd), Half Maximal Effective Concentration (EC50)
=, =0.36, 0.27 (0.24-0.30)
nM; nM
Antibody Function Kinetic measurements for the anti-TF antibodies,antibody affinity of 25A3.
Antibody Antigen Binding Assay Biacore-based measurements. The antibody was covalently coupled to a chip using an amine-coupling kit and a five-point titration of TF-His was analyzed using recommend procedures (GE Healthcare Bio-Sciences). Kinetic data were analyzed and fitted globally using a 1:1 binding model.
Antibody Activity Information 12 [1]
Activated Factor X Generation Percent (FXA)
100
%
A431 cells CVCL_0037 
Antigen Expression Positive Tissue factor expression (TF+++/++; 380,000 TF receptor copy number)
Antibody Function Evaluate competition between FVII and the human antibody(25A3) against TF.
Antibody Antigen Binding Assay TF-positive A431 cells were first incubated for 1 hour on ice with a titration of the human antibodies against TF or an IgG1 isotype control. Subsequently, FVII-Fc conjugated to A488 was added to the antibodycell mixture at a final concentration of 20 nmol/L. After another 1-hour incubation on ice, cells were washed, stained with a viability dye, and analyzed by flow cytometry. The A488 fluorescence data from viable cells was summarized using MFI. FVII-Fc binding was summarized with % FVII binding = [MFIanti-TF antibody labeled cells MFIunstainedcells]/[MFIIgG1 control labeled cells MFIunstainedcells].
Antibody Activity Information 13 [1]
Antibody Function Naked antibody (25A3) induces poor anticancer activity in PDX models with heterogeneous tissue factor expression.
Antibody Antigen Binding Assay TF-positive patient-derived xenograft (PDX) models were performed in athymic nude mice to evaluate the efficacy of the antibody in vivo.Study animals were implanted unilaterally on the left flank with tumor fragments. Animals were randomized and treated as indicated in the figures. Animals were removed from study and euthanized once tumor size reached 1,200 mm3 or skin ulceration was evident. In addition, the MTV curve for the treatment group in question was no longer shown once an animal was removed from study due to size. Antibody treatment started on day 1 after animals with a tumor size of approximately 190 mm3. The model dosed weekly at 10 mg/kg for 2 weeks.
Each Antibody-drug Conjugate Related to This Antibody
Full Information of The Activity Data of The ADC(s) Related to This Antibody
25A3-Val-Cit-MMAE [Investigative]
 ADC Info 
Discovered Using Cell Line-derived Xenograft Model
Click To Hide/Show 4 Activity Data Related to This Level
Experiment 1 Reporting the Activity Date of This ADC [1]
Efficacy Data Tumor Growth Inhibition value (TGI)
68.00% (Day 49)
Positive Tissue factor expression (TF+++/++; 570,000 TF receptor copy number)
Method Description
Cell line-derived xenograft (CDX) models MDA-MB-231 epidermoid carcinoma and the HPAF-II pancreatic carcinoma cell lines were implanted subcutaneously in the flank of athymic nude mice Animals were removed from study and euthanized once tumor size reached 1200 mm3 or skin ulceration was evident ADC was dosed weekly at 2 mg/kg for 2 weeks.

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In Vivo Model MDA-MB-231 cell line xenograft model
In Vitro Model Breast adenocarcinoma MDA-MB-231 cells CVCL_0062
Experiment 2 Reporting the Activity Date of This ADC [1]
Efficacy Data Tumor Growth Inhibition value (TGI)
100.00% (Day 49)
Low FOLR1 expression (FOLR1+)
Method Description
Cell line-derived xenograft (CDX) models MDA-MB-231 epidermoid carcinoma and the HPAF-II pancreatic carcinoma cell lines were implanted subcutaneously in the flank of athymic nude mice Animals were removed from study and euthanized once tumor size reached 1200 mm3 or skin ulceration was evident ADC was dosed weekly at 4 mg/kg for 2 weeks.

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In Vivo Model MDA-MB-231 cell line xenograft model
In Vitro Model Breast adenocarcinoma MDA-MB-231 cells CVCL_0062
Experiment 3 Reporting the Activity Date of This ADC [1]
Efficacy Data Tumor Growth Inhibition value (TGI)
100.00% (Day 39)
Low FOLR1 expression (FOLR1+)
Method Description
Cell line-derived xenograft (CDX) models The A431 epidermoid carcinoma and the HPAF-II pancreatic carcinoma cell lines were implanted subcutaneously in the flank of athymic nude mice Animals were removed from study and euthanized once tumor size reached 1200 mm3 or skin ulceration was evident ADC was dosed weekly at 2 mg/kg for 2 weeks.
In Vivo Model HPAF-II xenograft model
In Vitro Model Pancreatic ductal adenocarcinoma HPAF-II cells CVCL_0313
Experiment 4 Reporting the Activity Date of This ADC [1]
Efficacy Data Half Maximal Effective Concentration (EC50)
24.00 nM
Positive Tissue factor expression (TF+++/++; 380,000 TF receptor copy number)
Method Description
Antibody-dependent cellular cytotoxicity (ADCC) A431 cells were plated on a microtiter plate The following day, the cells were incubated with a ten-point 1:3 dilution titration of anti-TF antibodies or the ADCs starting at 50 nM An ADCC effector-to-target cell ratio of 8:1 was added to each well and incubated for 6 h at 37°C Luciferase Assay Reagent was added to each well to measure luminescence on an Envision plate reader Antibody-dependent cellular cytotoxicity (ADCC) reporter luminescence was evaluated after a 6-hour incubation of the reporter Jurkat cell line with TF-positive A431 cells and a titration of anti-TF antibody or ADC The ADCC reporter luminescence EC50 values for each anti-TF antibody or ADC are listed.

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In Vitro Model Human papillomavirus-related endocervical adenocarcinoma KB cells CVCL_0372
Revealed Based on the Cell Line Data
Click To Hide/Show 5 Activity Data Related to This Level
Experiment 1 Reporting the Activity Date of This ADC [1]
Efficacy Data Half Maximal Inhibitory Concentration (IC50)
5.00 nM
Positive Tissue factor expression (TF+++/++; 320,000 TF receptor copy number)
Method Description
To evaluate ADC cytotoxicity, cells were plated in 384-well plates Anti-TF antibodies conjugated to MC-vc-PAB-MMAE were serially diluted as shown Plates were incubated for 3 days, followed by lysis in CTG assay reagent For each ADC, the IC50 and its associated 95% confidence interval (95% CI) were calculated Titrations of the TF-specific ADCs were added to HPAF-II cells, with a 72-hour incubationThis treatment resulted in efficacious cell killing.

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In Vitro Model Pancreatic ductal adenocarcinoma HPAF-II cells CVCL_0313
Experiment 2 Reporting the Activity Date of This ADC [1]
Efficacy Data Half Maximal Inhibitory Concentration (IC50)
11.00 nM
Positive Tissue factor expression (TF+++/++; 570,000 TF receptor copy number)
Method Description
To evaluate ADC cytotoxicity, cells were plated in 384-well plates Anti-TF antibodies conjugated to MC-vc-PAB-MMAE were serially diluted as shown Plates were incubated for 3 days, followed by lysis in CTG assay reagent For each ADC, the IC50 and its associated 95% confidence interval (95% CI) were calculated Titrations of the TF-specific ADCs were added to 5-day old cultures of MDA-MB-231 cells, with a 72-hour incubationThis treatment resulted in efficacious cell killing.

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In Vitro Model Breast adenocarcinoma MDA-MB-231 cells CVCL_0062
Experiment 3 Reporting the Activity Date of This ADC [1]
Efficacy Data Half Maximal Inhibitory Concentration (IC50)
12.00 nM
Positive Tissue factor expression (TF+++/++; 320,000 TF receptor copy number)
Method Description
A431 cells were pre-incubated for 30 min without or with 50 nM of FVIIa prior to the addition of an anti-TF ADC (25A3-vc-MMAE) titration After a 4 h incubation at 37°C, the FVIIa and ADC were washed out and the cells were cultured for another 68 h before cell viability assessment.
In Vitro Model Skin squamous cell carcinoma A431 cells CVCL_0037
Experiment 4 Reporting the Activity Date of This ADC [2]
Efficacy Data Half Maximal Effective Concentration (EC50)
14.00 nM
Positive Tissue factor expression (TF+++/++; 320,000 TF receptor copy number)
Method Description
To evaluate ADC cytotoxicity, cells were plated in 384-well plates Anti-TF antibodies conjugated to MC-vc-PAB-MMAE were serially diluted as shown Plates were incubated for 3 days, followed by lysis in CTG assay reagent For each ADC, the IC50 and its associated 95% confidence interval (95% CI) were calculated Titrations of the TF-specific ADCs were added to A431 cells, with a 72-hour incubationThis treatment resulted in efficacious cell killing.

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In Vitro Model Skin squamous cell carcinoma A431 cells CVCL_0037
Experiment 5 Reporting the Activity Date of This ADC [2]
Efficacy Data Half Maximal Effective Concentration (EC50)
14.00 nM
Positive Tissue factor expression (TF+++/++; 320,000 TF receptor copy number)
Method Description
To evaluate ADC cytotoxicity, cells were plated in 384-well plates Anti-TF antibodies conjugated to MC-vc-PAB-MMAE were serially diluted as shown Plates were incubated for 3 days, followed by lysis in CTG assay reagent For each ADC, the IC50 and its associated 95% confidence interval (95% CI) were calculated Titrations of the TF-specific ADCs were added to A431 cells, with a 4-hour incubation followed by removal of excess ADC and culture for another 68 hours This treatment resulted in efficacious cell killing.

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In Vitro Model Skin squamous cell carcinoma A431 cells CVCL_0037
References
Ref 1 Treating Tissue Factor-Positive Cancers with Antibody-Drug Conjugates That Do Not Affect Blood Clotting. Mol Cancer Ther. 2018 Nov;17(11):2412-2426.
Ref 2 Discovery of STRO-002, a Novel Homogeneous ADC Targeting Folate Receptor Alpha, for the Treatment of Ovarian and Endometrial Cancers. Mol Cancer Ther. 2023 Feb 1;22(2):155-167.

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