This document summarizes research on developing a method for targeted delivery of interleukin-12 (IL-12) to tumors to enhance its anti-tumor effects while reducing systemic toxicity. Key points:
1) Separate administration and vascular targeting of the individual IL-12 subunits p35 and p40 could allow for their reassembly and activity specifically in tumors, avoiding toxicity from cytokine release syndrome.
2) Mutations were introduced to the subunits to allow their purification via different tags after co-expression and cleavage with TEV protease.
3) Surface plasmon resonance analysis showed the mutated subunits can still assemble into IL-12, and in vitro bioassays demonstrated the assembled IL-12 induces IFN
Much research aims tounderstand the cardiovascular system during disease development with angiogenesis, vasculature, atherosclerosis and heart research. Elabscience has 1200+ Antibodies for cardiovascular research. Worth to check!
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Accessing genetically tagged heterocycle libraries via a chemoresistant DNA s...Laura Berry
Presented at the Global Medicinal Chemistry and GPCR Summit. To find out more, visit:
www.global-engage.com
Andreas Brunschweiger, an Independent Group Leader at TU Dortmund, discusses the limitations of DNA-encoded compound libraries (DELs) and getting around these.
Los días 20 y 21 de octubre de 2016, la Fundacion Ramón Areces organizó un simposio internacional para analizar las 'Enfermedades raras de la piel: de la clínica al gen y viceversa'. El doctor Fernando Larcher Laguzzi, del CIEMAT-Universidad Carlos III de Madrid-IIS Fundación Jiménez Díaz, ejerció de coordinador.
Much research aims tounderstand the cardiovascular system during disease development with angiogenesis, vasculature, atherosclerosis and heart research. Elabscience has 1200+ Antibodies for cardiovascular research. Worth to check!
More antibodies click here:
http://www.elabscience.com/index.php/product/show/tid/73
Accessing genetically tagged heterocycle libraries via a chemoresistant DNA s...Laura Berry
Presented at the Global Medicinal Chemistry and GPCR Summit. To find out more, visit:
www.global-engage.com
Andreas Brunschweiger, an Independent Group Leader at TU Dortmund, discusses the limitations of DNA-encoded compound libraries (DELs) and getting around these.
Los días 20 y 21 de octubre de 2016, la Fundacion Ramón Areces organizó un simposio internacional para analizar las 'Enfermedades raras de la piel: de la clínica al gen y viceversa'. El doctor Fernando Larcher Laguzzi, del CIEMAT-Universidad Carlos III de Madrid-IIS Fundación Jiménez Díaz, ejerció de coordinador.
Sima Lev: VAP-B and its role in Amyotrophic lateral sclerosis Sima Lev
Amyotrophic lateral sclerosis (ALS) - Rapidly progressive neurodegenerative disorder characterized by death of motor neurons in the cerebral cortex, brain stem, and spinal cord.
Recombinant Expression and Purification of Aedes aegypti Midgut Serine Protea...Kamille Parungao
The Aedes aegypti mosquito is a major vector of blood-borne pathogens, such as the Dengue, Chikungunya, yellow fever, and Zika viruses. This poster discusses the recombinant expression and purification of a late-phase trypsin- like protease, Aedes aegypti serine protease VII (AaSPVII).
Sima Lev: VAP-B and its role in Amyotrophic lateral sclerosis Sima Lev
Amyotrophic lateral sclerosis (ALS) - Rapidly progressive neurodegenerative disorder characterized by death of motor neurons in the cerebral cortex, brain stem, and spinal cord.
Recombinant Expression and Purification of Aedes aegypti Midgut Serine Protea...Kamille Parungao
The Aedes aegypti mosquito is a major vector of blood-borne pathogens, such as the Dengue, Chikungunya, yellow fever, and Zika viruses. This poster discusses the recombinant expression and purification of a late-phase trypsin- like protease, Aedes aegypti serine protease VII (AaSPVII).
U.D. NACIMIENTO DE LA POLIFONÍA . ESTA BREVE U.D. TRATA DE LAS PRINCIPALES MANIFESTACIONES Y FUENTES DE LA POLIFONIA . ESTOS CONTENIDOS ESTÁS ADAPTADOS A ALUMNOS QUE ESTÁN CURSANDO 3º ESO EN LA MATERIA DE MÚSICA
Công ty tổ chức sự kiện VietSky - Năng lực thi công sự kiện
Công ty tổ chức sự kiện VietSky là công ty tổ chức sự kiện chuyên nghiệp hàng đầu tại Tp.HCM
Chuyên tổ chức các sự kiện và cung cấp các dịch vụ như:
- Tổ chức lễ khai trương: http://sukienvietsky.com/khai-truong-cong-ty-d-1037
- Tổ chức lễ khởi công: http://sukienvietsky.com/to-chuc-le-khoi-cong-dong-tho-d-1065
- Tổ chức hội thảo, hội nghị: http://sukienvietsky.com/hoi-nghi-khach-hang-to-chuc-hoi-nghi-hoi-thao-tai-hcm-d-1124
- Tổ chức lễ ra mắt sản phẩm: http://sukienvietsky.com/to-chuc-le-ra-mat-san-pham-moi-d-1066
- Tổ chức lễ kỹ niệm thành lập: http://sukienvietsky.com/cong-ty-to-chuc-le-ky-niem-thanh-lap-chuyen-nghiep-nhat-tai-tphcm-d-1552
- Tổ chức tiệc Galadinner: http://sukienvietsky.com/to-chuc-tiec-tat-nien-chuyen-nghiep-uy-tin-nhat-tai-tphcm-n-3281
- Tổ chức tiệc tất niên: http://sukienvietsky.com/to-chuc-tiec-tat-nien-chuyen-nghiep-uy-tin-nhat-tai-tphcm-n-3281
- Tổ chức lễ hội, Festival: http://sukienvietsky.com/to-chuc-le-hoi-van-hoa-ngay-hoi-truyen-thong-chuyen-nghiep-tai-tphcm-va-cac-tinh-thanh-n-3284
- Cung cấp PG chuyên nghiệp: http://sukienvietsky.com/cung-cap-pg-chuyen-nghiep-tai-tphcm-d-1039
- Cung cấp MC chuyên nghiệp: http://sukienvietsky.com/cung-cap-mc-chuyen-nghiep-tai-tphcm-d-1063
- Cung cấp Vũ đoàn, nhóm múa chuyên nghiệp: http://sukienvietsky.com/cung-cap-vu-doan-chuyen-nghiep-tai-hcm-d-1067
- Cung cấp ban nhạc Flamenco, nhóm nhạc: http://sukienvietsky.com/ban-nhac-nhac-cong-cho-thue-ban-nhac-chuyen-nghiep-tai-tphcm-n-3192
- Cung cấp ca sỹ chuyên nghiệp: http://sukienvietsky.com/ban-nhac-nhac-cong-cho-thue-ban-nhac-chuyen-nghiep-tai-tphcm-n-3192
- Cho thuê nhà bạt không gian: http://sukienvietsky.com/xuong-san-xuat-nha-bat-khong-gian-nha-leu-tai-tphcm-n-3301
- Cho thuê âm thanh chuyên nghiệp: http://sukienvietsky.com/am-thanh-anh-sang-n-3202
- Cho thuê ánh sáng chuyên nghiệp: http://sukienvietsky.com/am-thanh-anh-sang-n-3202
- Cho thuê màn hình LED chuyên nghiệp: http://sukienvietsky.com/cho-thue-man-hinh-led-chuyen-nghiep-gia-re-tai-tphcm-d-1638
- Cho thuê sân khấu chuyên nghiệp: http://sukienvietsky.com/cho-thue-san-khau-gia-re-chuyen-nghiep-tai-hcm-n-3288
- Cho thuê bàn, ghế chuyên nghiệp: http://sukienvietsky.com/thue-ban-ghe-cho-thue-ban-ghe-tai-hcm-n-3203
Tổ chức sự kiện trọn gói tại Tp.HCM và các tỉnh thành lân cận như: Tây Ninh, Bình Dương, Đồng Nai, Vũng Tàu, Bình Phước, Bình Thuận, Ninh Thuận, Nha Trang, Long An, Bến Tre, Tiền Giang, Vĩnh Long, Đồng Tháp, Cần Thơ, Hậu Giang, ….
Jugamos y aprendemos a convivir.
Descarga sesiones de aprendizaje. http://www.minedu.gob.pe/rutas-del-aprendizaje/sesiones2016/unidad1/segundogrado-edufisica.php
Cho thuê mc dẫn chương trình tiếng hoa chuyên nghiệp tại tp.hcm
Defense of thesis presentation
1. Activity on demand for Interleukin-12: !
A Holy Grail in the field of cancer immunotherapy!
Possible innovative strategies!
!
MSc Thesis defense of Danil Koovely !
Supervision of Dario Venetz during Spring Semester 2015!
Biomacromolecules research group of Prof. Dr. Dario Neri!
ETHZ!
3. Intermolecular disulfide bridge!
p35!
p40!
- IL-12 covalent heterodimer
composed of subunits p35 and
p40!
- Subunits expressed by separate
genes. p35 secretion is p40-
dependent.!
!
!
- One intermolecular disulfide
bridge present but not crucial for
IL-12 formation!
Structure of Interleukin-12!
4. 0 12 24 36 48 60 72
0
2
4
6
8
10
time [h]
%ID/g
blood
tumor
inactive p35
inactive p40
active IL-12 heterodimer
Main concept!
!
• Separate administration of individual subunits could prevent systemic toxicity!
• Vascular targeting could enable site-specific reassembly!
time gap
0 12 24 36 48 60 72
0
2
4
6
8
10
time [h]
%ID/g
blood
tumor
inactive p35
inactive p40
active IL-12 heterodimer
IL-12 activity restricted !
to the tumor site!
>> potentially no side-effects!
or!
or!
5. TEV protease!
!
24 h incubation at 4°C!
TEV
cleavage
site
p35*-‐scFv(F8)
p40*
Isolated
p35*-‐scFv(F8)
Aim: To separate p35*-scFv(F8) from p40* and TEV protease!
Method: To assess whether IL-12* can be cleaved by TEV and
purified by a “tandem” of IMAC and protein A purification!
TEV protease-mediated expression of p35-scFv(F8)!
I
M
A
C
p40*
+
p35*-‐scFv(F8)
+
TEV
TEV
Prot
A
p40*
+
p35*-‐scFv(F8)
p40*
p35*-‐scFv(F8)
6. 185
80
115
65
50
30
25
15
10
TEV
IL-‐12*
NR
R
NR
R
L
NR
R
IL-‐12*+TEV
Non cleaved IL-12*!
p35*-scFv(F8)!
p40*!
TEV protease!
à The cleavage site is accessible by the TEV protease!
à IL-12*-TEV can be split into p35*-scFv and p40*!
IL-12* and TEV protease were
mixed in a molar ratio 1:1 and
incubated at 4°C overnight!
7.
L
I
FT
W
E
FT
W
E
185
80
115
65
50
30
25
15
10
A
B
C
D
E
L: Ladder!
I: Input!
Ft: Flow-through IMAC !
W: Wash IMAC !
E: Eluate IMAC !
Ft: Flow-through Protein A !
W: Wash Protein A !
E: Concentrated eluate
Protein A!
A: Non-cleaved IL-12*
aggregates!
B: Non-cleaved IL-12*!
C: Cleaved p35*-scFv(F8)!
D: Cleaved p40*!
E: TEV enzyme!
IMAC
Protein
A
à Both IL-12* subunits are “sticky” !
à Despite successful TEV cleavage it is difficult to efficiently separate p35* from p40*!
8. Co-expression of mutated IL-12 subunits !
p35*-‐tag
p40*-‐tag
L
NR
R
NR
R
NR
R
185
80
115
65
50
30
25
15
10
185
80
115
65
50
30
25
15
10
p35*-‐Db(F8)
p40*-‐Db(F8)
p35*-‐His
p40*-‐His
p35*-‐scFv(F8)
p40*-‐scFv(F8)
L
NR
R
NR
R
NR
R
I
M
A
C
Prot
A
+
1 to 2 molar ratio!
13. 5 10 15 20
0
5
10
15
20
25
Positive control S200 10−300
Volume [mL]
mAU
5 10 15 20
0
5
10
15
20
25
p40−scFv(F8) S200 10−300
Volume [mL]
mAU
5 10 15 20
0
5
10
15
20
25
p35−scFv(F8) S200 10−300
Volume [mL]
mAU
14.1
mL
12.9
mL
12.2
mL
10.9
mL
5 10 15 20
0
5
10
15
20
25
p40/p35 (scFV) 1:1 mol ratio
Volume [mL]
mAU
5 10 15 20
0
5
10
15
20
25
p40/p35 1:2 (scFv) mol ratio
Volume [mL]
mAU
5 10 15 20
0
5
10
15
20
25
p40/p35 1:4 (scFv) mol ratio
Volume [mL]
mAU
FPLC confirmation of in vitro assembly!
14. Summary and outlook
5 10 15 20
0
5
10
15
20
25
Positive control S200 10−300
Volume [mL]
mAU
5 10 15 20
0
5
10
15
20
25
p40−scFv(F8) S200 10−300
Volume [mL]
mAU
5 10 15 20
0
5
10
15
20
25
p35−scFv(F8) S200 10−300
Volume [mL]
mAU
Alternative strategy for p35 production through TEV cleavage was tested and
found to be not implementable!
!
Different p35-antibody constructs were produced with the co-transfection
strategy and successfully isolated. To our knowledge p35 has never been
isolated in such a pure quality !
!
p35- and p40-antibody constructs were found to bind readily in SPR
measurements but not so well in SEC. Biological activity in vitro of these
constructs was checked and confirmed!
!
!
!
!
!
Behavior in vitro has to be analyzed further in order to explain the different
results of FPLC and bioassay!
!
Behavior of p35-antibody constructs in vivo has to be analyzed in a
biodistribution study!
!