Antibody-drug Conjugate Information

General Information of This Antibody-drug Conjugate (ADC)
ADC ID
DRG0ABIGI
ADC Name
wO2025019776A2ADC 52-8
Synonyms
WO2025019776A2ADC 52-8
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Organization
EXELIXIS, INC.
Drug Status
Investigative
Drug-to-Antibody Ratio
8
Structure
Antibody Name
A52
  Antibody Info 
Antigen Name
Interleukin-13 receptor subunit alpha-2 (IL13RA2)
  Antigen Info 
Payload Name
Belotecan
  Payload Info 
Therapeutic Target
DNA topoisomerase 1 (TOP1)
  Target Info 
Linker Name
WO2025019776A2, ADC 22-8, Linker
  Linker Info 
Conjugate Type
site formylglycine residue random conjugation
Combination Type
IIa
ADC-specific functional property(2027 Update)
Bystander Killing Effect
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Bystander Killing Effect Description Reference
yes
The potential effects of ADC 52-8 on human myeloid (CFU-GM) was assessed using colony forming cell assays. The colonies were divided into the following categories based on size and morphology: Erythroid (CFU-E and BFU-E),myeloid (CFU-GM),and multi-potential (CFU- GEMM).Photographs were taken of representative hematopoietic progenitor-derived.

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[1]
Payload Release Efficiency
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Incubation Time 14day Release 21.85ug/mL Reference
[1]
Description
Clonogenic progenitors of human myeloid and erythroid progenitors were assessed in a semi-solid methylcellulose-based media formulation containing 35%FBS,2%BSA, rhIL-3 (10 ng/mL),rhGM-CSF (10 ng/mL),rhSCF (50 ng/mL)and Epo (3 U/mL).The tested ADCs were added in at various concentrations and the tubes were vortexed to ensure equal distribution of the ADC throughout the matrix.5-FU (Sigma Aldrich)was used as a positive control for progenitor proliferation (inhibition of colony growth)and was introduced to the bone marrow cultures at 1.0,0.1,and 0.01ug/mL.The assays were initiated with bone marrow mononuclear cells (BMMNC)at 2x104cells per culture.Following 14 days in culture,myeloid and erythroid colonies were assessed microscopically and scored by trained personnel.The colonies were divided into the following categories based on size and morphology:Erythroid (CFU-E and BFU-E),myeloid (CFU-GM),and multi-potential (CFU- GEMM).Photographs were taken of representative hematopoietic progenitor-derived colonies from various lineages,illustrating colonies in the presence of the solvent control as well as colonies in the presence of the test ADCs.

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Incubation Time 14day Release 5.88ug/mL Reference
[1]
Description
Clonogenic progenitors of human myeloid and erythroid progenitors were assessed in a semi-solid methylcellulose-based media formulation containing 35%FBS,2%BSA, rhIL-3 (10 ng/mL),rhGM-CSF (10 ng/mL),rhSCF (50 ng/mL)and Epo (3 U/mL).The tested ADCs were added in at various concentrations and the tubes were vortexed to ensure equal distribution of the ADC throughout the matrix.5-FU (Sigma Aldrich)was used as a positive control for progenitor proliferation (inhibition of colony growth)and was introduced to the bone marrow cultures at 1.0,0.1,and 0.01ug/mL.The assays were initiated with bone marrow mononuclear cells (BMMNC)at 2x104cells per culture.Following 14 days in culture,myeloid and erythroid colonies were assessed microscopically and scored by trained personnel.The colonies were divided into the following categories based on size and morphology:Erythroid (CFU-E and BFU-E),myeloid (CFU-GM),and multi-potential (CFU- GEMM).Photographs were taken of representative hematopoietic progenitor-derived colonies from various lineages,illustrating colonies in the presence of the solvent control as well as colonies in the presence of the test ADCs.

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Binding Affinity
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Dissocation Constant (Kd) Binding Target Description Reference
0.2891 nM
hIL13Ra2
LISA was performed to assess the binding profiles of the ADCs produced as described above and their parental antibodies to human IL13Ra2-His protein.
[1]
Circulating Stability
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Incubation Time 11day Release 0.54ug/mL Reference
[1]
Incubation Medium In the neutrophil assay,CD34 cells were thawed,washed,and counted. A cell stock was prepared in the initial media formulation containing X-Vivo-15,rhIL-3 (20 ng/mL),rhIL-6 (20 ng/mL),rhSCF (100 ng/mL)and rhFlt-3L (100 ng/mL).Approximately 10,000 cells in 200 uL volume were plated per well in round-bottom 96-well plates and cultured in a humidified incubator at 37°C,5%CO2for 3 days.After this time,the cells were washed twice with X-Vivo medium and incubated in X-Vivo-15 medium supplemented with rhSCF (50 ng/mL),rhFlt-3L (100 ng/mL),rh IL3 (5 ng/mL),rhGM-CSF (5ng/mL)rhG-CSF (5 ng/mL)for 4 days.The cells were again washed (x2)and incubated in X-Vivo-15 medium supplemented with rhIL-3 (5 ng/mL)and rhG-CSF (30 ng/mL)for another 4 days.On day 11,the cells were washed twice and placed in the final media formulation containing X-Vivo- 15 supplemented with just rhG-CSF (30 ng/mL)for the final 6 days.The test articles were added at this stage and were retained in the assay for the final 6 days.The effect of ADCs on neutrophil differentiation was represented by the percentage and number (events)of CD66b within the viable cell population. The concentration of 50%inhibition (ICso)was calculated based on the sigmoid curve fit using Dose-Response,One-Site Model formula:y=A+[ (B- A)/ (1+ ((C/x)^D)),where A=the initial value (baseline response),B=maximum response,C=center (drug concentration that provokes a response halfway between A and B) and D=slope of the curve at midpoint.
Description
The stability of ADC linkers were assessed using a neutrophil assay. The effect of ADCs on neutrophil differentiation was represented by the percentage and number (events)of CD66b within the viable cell population.

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General Information of The ADMET Data Related to This ADC(2027 Update)
Absorption
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Standard Type Value Units Description Reference
Area Under the Concentration-Time Curve (AUC) ≈6000000 hr&#42ng/mL
Male JVC/FVC cannulated Sprague-Dawley rats (5 per group)were dosed intravenously with a single dose of 5mg/kg of the tested ADC on Day 1 after 16 hours of fasting.100uL K2EDTA plasma was collected at 30 min,4h,24h (on Day 2),168h (on Day 8),240h (on Day 11),336h (on Day 15),and 504h (on Day 22)post-dose,saved in bullet tubes and stored at -25°C until end of the study.

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 [1]
Distribution
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Standard Type Value Units Description Reference
Area Under the Concentration-Time Curve (AUC) ≈6000000 hr&#42ng/mL
Male JVC/FVC cannulated Sprague-Dawley rats (5 per group)were dosed intravenously with a single dose of 5mg/kg of the tested ADC on Day 1 after 16 hours of fasting.100uL K2EDTA plasma was collected at 30 min,4h,24h (on Day 2),168h (on Day 8),240h (on Day 11),336h (on Day 15),and 504h (on Day 22)post-dose,saved in bullet tubes and stored at -25°C until end of the study.

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 [1]
Metabolism
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Standard Type Value Units Description Reference
Area Under the Concentration-Time Curve (AUC) ≈6000000 hr&#42ng/mL
Male JVC/FVC cannulated Sprague-Dawley rats (5 per group)were dosed intravenously with a single dose of 5mg/kg of the tested ADC on Day 1 after 16 hours of fasting.100uL K2EDTA plasma was collected at 30 min,4h,24h (on Day 2),168h (on Day 8),240h (on Day 11),336h (on Day 15),and 504h (on Day 22)post-dose,saved in bullet tubes and stored at -25°C until end of the study.

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 [1]
Excretion
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Standard Type Value Units Description Reference
Area Under the Concentration-Time Curve (AUC) ≈6000000 hr&#42ng/mL
Male JVC/FVC cannulated Sprague-Dawley rats (5 per group)were dosed intravenously with a single dose of 5mg/kg of the tested ADC on Day 1 after 16 hours of fasting.100uL K2EDTA plasma was collected at 30 min,4h,24h (on Day 2),168h (on Day 8),240h (on Day 11),336h (on Day 15),and 504h (on Day 22)post-dose,saved in bullet tubes and stored at -25°C until end of the study.

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 [1]
Clearance (CL) ≈0.7 mL/hr/kg
Male JVC/FVC cannulated Sprague-Dawley rats (5 per group)were dosed intravenously with a single dose of 5mg/kg of the tested ADC on Day 1 after 16 hours of fasting.100uL K2EDTA plasma was collected at 30 min,4h,24h (on Day 2),168h (on Day 8),240h (on Day 11),336h (on Day 15),and 504h (on Day 22)post-dose,saved in bullet tubes and stored at -25°C until end of the study.

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 [1]
Elimination Half-Life (t1/2) ≈210 hr
Male JVC/FVC cannulated Sprague-Dawley rats (5 per group)were dosed intravenously with a single dose of 5mg/kg of the tested ADC on Day 1 after 16 hours of fasting.100uL K2EDTA plasma was collected at 30 min,4h,24h (on Day 2),168h (on Day 8),240h (on Day 11),336h (on Day 15),and 504h (on Day 22)post-dose,saved in bullet tubes and stored at -25°C until end of the study.

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 [1]
General Information of The Activity Data Related to This ADC
Discovered Using Cell Line-derived Xenograft Model
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Standard Type Value Units Cell Line Disease Model
Tumor Growth lnhibition value (TGl) 
66.6
%
Undisclosed Undisclosed
Tumor Growth lnhibition value (TGl) 
83.4
%
Undisclosed Undisclosed
Revealed Based on the Cell Line Data
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Standard Type Value Units Cell Line Disease Model
the MFI (geometric mean) signal 
≈ 30000-40000
.
CVCL_0045
Normal
the MFI (geometric mean) signal 
≈ 40000-50000
.
CVCL_0132
Amelanotic melanoma
Half Maximal inhibitory Concentration (lC50) 
2
nM
CVCL_0132
Amelanotic melanoma
Half Maximal inhibitory Concentration (lC50) 
> 100
nM
CVCL_1495
Lung adenocarcinoma
Half Maximal inhibitory Concentration (lC50) 
> 100
nM
CVCL_1543
Lung adenocarcinoma
Half Maximal inhibitory Concentration (lC50) 
> 100
nM
CVCL_0630
Lung squamous cell carcinoma
Full List of Activity Data of This Antibody-drug Conjugate
Discovered Using Cell Line-derived Xenograft Model
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Experiment 1 Reporting the Activity Date of This ADC [1]
Efficacy Data Tumor Growth lnhibition value (TGl) 66.60% Positive IL13Ra2 expression (IL13Ra2+++/++)
Method Description
Each female BALB/c Nude mouse was inoculated subcutaneously in the right upper/lower flank region with 1x10 7of H2228 tumor cells (an NSCLC cell line having an IL13Ra2 copy number of about 2,000)in 0.1 mL of PBS mixed with MATRIGELR (1:1)for tumor development.When the mean tumor size reached about 80-150 mm 3,mice were randomized into respective treatment groups (10 mice per group)and received intravenous injections of vehicle or a single dose of the tested ADCs at 10 mg/kg on Day 1. Body weights and tumor volumes were measured twice per week until the end of the study (Day 48).

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In Vivo Model CDX Model-H2228
Experiment 2 Reporting the Activity Date of This ADC [1]
Efficacy Data Tumor Growth lnhibition value (TGl) 83.40% Positive IL13Ra2 expression (IL13Ra2+++/++)
Method Description
Each female BALB/c Nude mouse was inoculated subcutaneously in the right upper flank region with 0.5x10 6of SK-MES-1 tumor cells (a non-small cell lung cancer (NSCLC)cell line having an IL13Ra2 copy number of about 4x104)in 0.1 mL of PBS for tumor development.When the mean tumor size reached about 80-150 mm 3,mice were randomized into respective treatment groups (10 mice per group)and received intravenous injections of PBS vehicle or a single dose of the tested ADCs at 10 mg/kg on Day 1. The end of the study was day 49.

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In Vivo Model CDX Model-SK-MES-1
Revealed Based on the Cell Line Data
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Experiment 1 Reporting the Activity Date of This ADC [1]
Efficacy Data the MFI (geometric mean) signal ≈ 30000-40000 . Positive hIL13Ra2 expression (hIL13Ra2+++/++)
Method Description
FACS was used to evaluate the binding profiles of HEK293 cells engineered to express IL13Ra2 (HEK293-IL13Ra2) cells expressing IL13Ra2. The samples were then read by flow cytometry and the MFI (geometric mean) signal was calculated.
In Vitro Model Normal HEK293 cells CVCL_0045
Experiment 2 Reporting the Activity Date of This ADC [1]
Efficacy Data the MFI (geometric mean) signal ≈ 40000-50000 . Positive hIL13Ra2 expression (hIL13Ra2+++/++)
Method Description
FACS was used to evaluate the binding profiles of A375 cells expressing IL13Ra2. The samples were then read by flow cytometry and the MFI (geometric mean) signal was calculated.
In Vitro Model Amelanotic melanoma A375 cells CVCL_0132
Experiment 3 Reporting the Activity Date of This ADC [1]
Efficacy Data Half Maximal inhibitory Concentration (lC50) 2 nM High hIL13Ra2 expression (hIL13Ra2 +++)
Method Description
The cytotoxic activity of ADC 52-8 was determined against cancer cell lines A375.FITC and anti-Hen egg-white lysozyme isotype control antibody (HEWL)conjugated to the same linker payloads.
In Vitro Model Amelanotic melanoma A375 cells CVCL_0132
Experiment 4 Reporting the Activity Date of This ADC [1]
Efficacy Data Half Maximal inhibitory Concentration (lC50) > 100 nM Positive hIL13Ra2 expression (hIL13Ra2+++/++)
Method Description
The cytotoxic activity of ADC 52-8 was determined against cancer cell lines H1792.FITC and anti-Hen egg-white lysozyme isotype control antibody (HEWL)conjugated to the same linker payloads.
In Vitro Model Lung adenocarcinoma H1792 cells CVCL_1495
Experiment 5 Reporting the Activity Date of This ADC [1]
Efficacy Data Half Maximal inhibitory Concentration (lC50) > 100 nM Positive hIL13Ra2 expression (hIL13Ra2+++/++)
Method Description
The cytotoxic activity of ADC 52-8 was determined against cancer cell lines H2228.FITC and anti-Hen egg-white lysozyme isotype control antibody (HEWL)conjugated to the same linker payloads.
In Vitro Model Lung adenocarcinoma H2228 cells CVCL_1543
Experiment 6 Reporting the Activity Date of This ADC [1]
Efficacy Data Half Maximal inhibitory Concentration (lC50) > 100 nM Positive hIL13Ra2 expression (hIL13Ra2+++/++)
Method Description
The cytotoxic activity of ADC 52-8 was determined against cancer cell lines SK-MES-1.FITC and anti-Hen egg-white lysozyme isotype control antibody (HEWL)conjugated to the same linker payloads.
In Vitro Model Lung squamous cell carcinoma SK-MES-1 cells CVCL_0630
References
Ref 1 Interleukin-13 receptor subunit alpha-2 antibody-drug conjugates and uses thereof