RESEARCH POSTER PRESENTATION DESIGN © 2015 www.PosterPresentations.com Antibody–drug conjugates (ADCs) are a powerful combination of biological and chemical drugs, that combine specific monoclonal antibodies (mAbs) with potent chemotherapeutic drugs. ADCs can kill tumor cells effectively without damaging normal cells by controlling the delivery of the intracellular toxin to tumor cells with the antigen-targeting specificity of mAbs. As a result, ADCs are rising in oncotherapy as antineoplastic drugs due to their decreased side effects. Here, we compare first generation ADCs that were manufactured in our hands and our second generation ADCs using our thiol-covalent conjugation method. Introduction First Generation ADCs In vivo antitumor activity of Abs, ADC and small molecule drugs in NCI-N87 xenograft models. (A) In vivo tumor inhibition effect of the first run treatment. ADC was given as a single intravenous injection at doses of 2.5, 5 or 10 mg/kg on day 0. Herceptin (10 mg/kg) was injected intravenously on days 0, 7, and 14. Lapatinib (200 mg/kg) was given daily through intragastric administration from day 0 to day 20. (B) In vivo tumor inhibition effect of the second run treatment. The effects of ADC (0.5 or 5 mg/kg), mAb (5 mg/kg), free MMAE (0.1 mg/kg equivalent of 5 mg/kg ADC) mono-treatment, and mAb at 5 mg/kg, MMAE at 0.1 mg/kg co-treatment were evaluated. The mice in all groups were administrated drug at days 0 and 7 after randomization. (C) Subcutaneous tumors in the first run treatment were measured at day 21. (D) Subcutaneous tumors in the second run treatment were measured at day 21. First Generation ADCs continued Second Generation ADCs continued References 1. Yao, X.; Jiang, J.; Wang, X.; Huang, C.; Li, D.; Xie, K.; Xu, Q.; Li, H.; Li, Z.; Lou, L.et al. Breast Cancer Research and Treatment 2015, 153 (1), 123. 2. Li, H.; Yu, C.; Jiang, J.; Huang, C.; Yao, X.; Xu, Q.; Yu, F.; Lou, L.; Fang, Cancer Biology & Therapy 2016, 17 (4), 346. 3. Huang, C., Fang J., Ye H., Zhang L., WO 2017031034 A3. Contacts E-mail: [email protected], [email protected] Acknowledgements："Major new drug discovery" major scientific and technological special project Molecular structure of antibody drug conjugate (ADC): mAb-Mc-VC- PAB-MMAE MabPlex International, Yantai City, Shandong Province, China Xuejing Yao, Hongwen Li, Hui Ye, Lele Li, Bethanne Deuel, Andrew C. Huang, Jianmin Fang The Development of a Next Generation Antibody-Drug Conjugate (ADC) Hydrophobic interaction chromatography (HIC) analysis of first generation ADC. SEC/MS of first generation ADC O N O O NH N O O O N O O NH OH O N H O H N HN O H 2 N O N H N O O S mAb Mc VC PAB Monomethyl auristatin E (MMAE) Site of hydrolysis by cathepsin B Second Generation ADCs d Screening of antibody drug conjugates Key Attributes mAb Concentrations Buffer, pH, Ions Sequence of Adding Mixing Speed & Method Reduction Temperatrure Reduction Time Reductant Anti-Oxydants mAb-Drug Ratio Catalysts Organic Solvents Conjugation Temperatures linker payload linker payload linker payload linker payload linker payload linker payload linker payload linker payload linker payload linker payload ADC 1: Losing two disulfide bond support. 26.7% ADC 2: Remaining support of disulfide bonds. 18.9% ADC 3: Coupling disulfide bonds with the linker. 5.1% Three types of ADCs and their elimination rate in humans ADC 3 is much more stable than the other 2 ADCs in human peripheral blood, which can reduce side effects in the systemic circulation. HIC analysis of a second generation ADC. Conjugation of Human IgG1 yields of DAR 4 can be 80%-90% consistently. Minutes 14 16 18 20 22 24 26 28 30 32 AU 0.00 0.01 0.02 0.03 0.04 0.05 AU 0.00 0.01 0.02 0.03 0.04 0.05 1 2 3 4 5 6 7 8 9 PDA - 220nm Pk # mAb SK-BR-3 -2 0 2 4 6 0 20 40 60 80 C15213-ADC-3 C15213-ADC-4 concentration(log ng/ml) in h ib it io n ADC-1 ADC-2 mAb SK-BR-3 ADC-1 ADC-2 IC 50 (ng/mL) 3.263 4.138 MIR (%) 80 80 R 2 0.9925 0.9975 Inhibitory Effects of two different Second generation ADCs vs. Naked mAb Inhibitory Effects of Human Tumor Cell Growth in Nude Mice After a Treatment Time of 21 Days. DAR 4 Conclusions MabPlex has successfully developed a first generation ADC method to treat malignant tumors, such as gastric cancer and breast cancer, which has exhibited potent anti-tumor effects both in vitro and in vivo. The production capacity of mAb and ADC has run up to 2000 L and 150 L respectively according to cGMP specifications meeting the USFDA, EMA and CFDA standards. Furthermore, this ADC can be stored stably for 36 months under -80 o C. Significantly, this ADC received the first clinical approval in China for human trials. Additionally, in order to increase homogenicity of the DAR and other key attributes, we cloned various novel mAbs and synthesized a variety of new linker-payloads for ADC screening. We have accomplished our conjugation process optimization of mAbs with linker-drugs based on the key attributes of the reduction and optimal conjugation of mAbs. Finally, we developed a platform for preparing a new generation of ADCs, which can distinctly increase the drug ability of naked mAbs. ADCs from this platform have markedly enhanced stability in the blood with demonstrably lower heterogenicity and good efficacy. Second Generation ADCs continued MabPlex consistently develops and manufactures first generation ADCs for clients with DAR4 of 50% using first generation ADC linkage methods. ISSUE:::While these first generation ADCs are efficacious as you can see above, first generation ADCs suffer from heterogenicity. It is believed that this heterogenicity decreases the drug ability of the ADC and also increases the toxicity of the ADC in the patient. Variable drug to antibody ratios (DAR) are also known to create variable efficacies. In a desire to create a more homogeneous ADC, we created our second generation ADC method using our patented thiol-covalent conjugation method. Treatment Dosage Tumor Volume (mm 2 ) Vehicle ________ 1084 ADC-1 10mg/kg 536 ADC-3 10mg/kg 450 ADC-4 1mg/kg 10
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RESEARCH POSTER PRESENTATION DESIGN © 2015
www.PosterPresentations.com
Antibody–drug conjugates (ADCs) are a powerful combination of biological
and chemical drugs, that combine specific monoclonal antibodies (mAbs) with
potent chemotherapeutic drugs. ADCs can kill tumor cells effectively without damaging normal cells by controlling the delivery of the intracellular toxin to
tumor cells with the antigen-targeting specificity of mAbs. As a result, ADCs
are rising in oncotherapy as antineoplastic drugs due to their decreased
side effects. Here, we compare first generation ADCs that were manufactured
in our hands and our second generation ADCs using our thiol-covalent
conjugation method.
Introduction
First Generation ADCs
In vivo antitumor activity of Abs, ADC and small molecule drugs in NCI-N87 xenograft models. (A) In vivo tumor inhibition effect of the first run treatment. ADC was given as a single intravenous injection at doses of 2.5, 5 or 10 mg/kg on day 0. Herceptin (10 mg/kg) was injected intravenously on days 0, 7, and 14. Lapatinib (200 mg/kg) was given daily through intragastric administration from day 0 to day 20. (B) In vivo tumor inhibition effect of the second run treatment. The effects of ADC (0.5 or 5 mg/kg), mAb (5 mg/kg), free MMAE (0.1 mg/kg equivalent of 5 mg/kg ADC) mono-treatment, and mAb at 5 mg/kg, MMAE at 0.1 mg/kg co-treatment were evaluated. The mice in all groups were administrated drug at days 0 and 7 after randomization. (C) Subcutaneous tumors in the first run treatment were measured at day 21. (D) Subcutaneous tumors in the second run treatment were measured at day 21.
First Generation ADCs continued Second Generation ADCs continued
References1. Yao, X.; Jiang, J.; Wang, X.; Huang, C.; Li, D.; Xie, K.; Xu, Q.; Li, H.; Li, Z.; Lou, L.et al. Breast Cancer Research and Treatment 2015, 153 (1), 123. 2. Li, H.; Yu, C.; Jiang, J.; Huang, C.; Yao, X.; Xu, Q.; Yu, F.; Lou, L.; Fang, Cancer Biology & Therapy 2016, 17 (4), 346. 3. Huang, C., Fang J., Ye H., Zhang L., WO 2017031034 A3.
Contacts
E-mail: [email protected], [email protected] Acknowledgements："Major new drug discovery" major scientific and technological special project
Molecular structure of antibody drug conjugate (ADC): mAb-Mc-VC-PAB-MMAE
MabPlex International, Yantai City, Shandong Province, ChinaXuejing Yao, Hongwen Li, Hui Ye, Lele Li, Bethanne Deuel, Andrew C. Huang, Jianmin Fang
The Development of a Next Generation Antibody-Drug Conjugate (ADC)
Hydrophobic interaction chromatography (HIC) analysis of first generation ADC.
SEC/MS of first generation ADC
O N
O
O
NH N
O
O O
NO O
NHOH
O
NH
O
HN
HN
OH2N
O
NH
N
O
O
S
mAb
Mc VC PAB Monomethyl auristatin E (MMAE)
Site of hydrolysisby cathepsin B
Second Generation ADCs
d
Screening of antibody drug conjugates
Key
Attributes
mAb Concentrations
Buffer, pH, Ions
Sequence of Adding
Mixing Speed & Method
Reduction Temperatrure
Reduction Time
Reductant
Anti-Oxydants
mAb-Drug Ratio
Catalysts
Organic Solvents
Conjugation Temperatures
linker
payload
linker
payload
linker
payload
linker
payloadlinker
payload
linker
payload
linker
payload
linker
payload
linker
payload
linker
payload
ADC 1: Losing two disulfide bond support.
26.7%
ADC 2: Remaining support of disulfide bonds.
18.9%
ADC 3: Coupling disulfide bonds with the linker.
5.1%
Three types of ADCs and their elimination rate in humans
ADC 3 is much more stable than the other 2 ADCs in human peripheral blood, which can reduce side effects in the systemic circulation.
HIC analysis of a second generation ADC. Conjugation of Human IgG1 yields of DAR 4 can be 80%-90% consistently.
Minutes14 16 18 20 22 24 26 28 30 32
AU
0.00
0.01
0.02
0.03
0.04
0.05
AU
0.00
0.01
0.02
0.03
0.04
0.05
1 2 3 45
6
7
8
9
PDA - 220nmPk #
mAb
S K -B R -3
-2 0 2 4 60
2 0
4 0
6 0
8 0
C 1 5 21 3 -A D C - 3C 1 5 21 3 -A D C - 4
co n c e n t r a t io n ( lo g n g /m l)
inh
ibit
ion
ADC-1
ADC-2
mAb
SK-BR-3 ADC-1 ADC-2
IC50(ng/mL) 3.263 4.138
MIR (%) 80 80
R2 0.9925 0.9975
Inhibitory Effects of two different Second generation ADCs vs. Naked mAb
Inhibitory Effects of Human Tumor Cell Growth in Nude Mice After a Treatment Time of 21 Days.
DAR 4
ConclusionsMabPlex has successfully developed a first generation ADC method to treat
malignant tumors, such as gastric cancer and breast cancer, which has exhibited
potent anti-tumor effects both in vitro and in vivo. The production capacity of
mAb and ADC has run up to 2000 L and 150 L respectively according to
cGMP specifications meeting the USFDA, EMA and CFDA standards.
Furthermore, this ADC can be stored stably for 36 months under -80oC.
Significantly, this ADC received the first clinical approval in China for human
trials.
Additionally, in order to increase homogenicity of the DAR and other key
attributes, we cloned various novel mAbs and synthesized a variety of new
linker-payloads for ADC screening. We have accomplished our conjugation
process optimization of mAbs with linker-drugs based on the key attributes of
the reduction and optimal conjugation of mAbs. Finally, we developed a
platform for preparing a new generation of ADCs, which can distinctly increase
the drug ability of naked mAbs. ADCs from this platform have markedly
enhanced stability in the blood with demonstrably lower heterogenicity and
good efficacy.
Second Generation ADCs continued
MabPlex consistently develops and manufactures first generation ADCs for
clients with DAR4 of 50% using first generation ADC linkage methods.
ISSUE:::While these first generation ADCs are efficacious as you can see
above, first generation ADCs suffer from heterogenicity. It is believed that this
heterogenicity decreases the drug ability of the ADC and also increases the
toxicity of the ADC in the patient. Variable drug to antibody ratios (DAR) are
also known to create variable efficacies. In a desire to create a more
homogeneous ADC, we created our second generation ADC method using our
patented thiol-covalent conjugation method.
Treatment Dosage Tumor Volume (mm2)
Vehicle ________ 1084
ADC-1 10mg/kg 536
ADC-3 10mg/kg 450
ADC-4 1mg/kg 10
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