Antibody Information

General Information of This Antibody
Antibody ID
ANI0NNOMT
Antibody Name
Anit-TF mAb 1F
Antibody Type
Monoclonal antibody (mAb)
Antibody Subtype
Human IgG1-kappa
Antigen Name
Tissue factor (F3)
  Antigen Info 
The Activity Data of This Antibody
Antibody Activity Information 1 [1]
Half Maximal Inhibitory Concentration (IC50)
0.07
nM
A431 cells CVCL_0037 
Antigen Expression Positive Tissue factor expression (TF+++/++; 380,000 TF receptor copy number)
Antibody Function TF-specific antibody(1F) 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 2 [1]
Half Maximal Inhibitory Concentration (IC50)
1
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 (1F) 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 3 [1]
Half Maximal Inhibitory Concentration (IC50)
1.3
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 (1F) 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 4 [1]
Half Maximal Effective Concentration (EC50)
0.05
nM
HCT 116 cells CVCL_0291 
Antigen Expression Positive Tissue factor expression (TF+++/++; 22,000 copies of surface)
Antibody Function Anti-TF antibody (1F) 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 5 [1]
Half Maximal Effective Concentration (EC50)
0.49
nM
A431 cells CVCL_0037 
Antigen Expression Positive Tissue factor expression (TF+++/++; 380,000 TF receptor copy number)
Antibody Function Anti-TF antibody (1F) 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 6 [1]
Half Maximal Effective Concentration (EC50)
1.5
nM
Antibody Function Kinetic measurements for the anti-TF antibodies,antibody affinity of 1F.
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 7 [1]
Dissociation Constant (Kd), Half Maximal Effective Concentration (EC50)
=, =0.31, 0.17 (0.14-0.21)
nM; nM
Antibody Function Kinetic measurements for the anti-TF antibodies,antibody affinity of 1F.
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 8 [1]
Activated Factor X Generation Percent (FXA)
35
%
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(1F) 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].
Each Antibody-drug Conjugate Related to This Antibody
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.

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