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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!
Boosted proliferation!
Increased !cytotoxicity!
IL-12!
APCs!
IFN-γ!
STRONG!ANTI-TUMOR!EFFECT!
High systemic toxicity due to Cytokine Release Syndrome limits clinical applications !
Introduction to Interleukin-12!
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!
0 12 24 36 48 60 720
2
4
6
8
10
time [h]
%ID
/g
bloodtumor
inactive p35inactive p40active 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 60 720
2
4
6
8
10
time [h]
%ID
/g
bloodtumor
inactive p35inactive p40active IL-12 heterodimer
IL-12 activity restricted !to the tumor site! >> potentially no side-effects!
or!
or!
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 MA C
p40* + p35*-‐scFv(F8) + TEV
TEV
Prot A
p40* + p35*-‐scFv(F8) p40*
p35*-‐scFv(F8)
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!
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*!
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 MA C
Prot A +
1 to 2 molar ratio!
p35-‐His No SEC due to small quanLty of protein
SEC purificaLon
Ready for further proteins characterization: !1) SPR for binding kinetics between p35* and p40*!2) Bioassay of pre-assembled IL-12*!3) SEC analysis of assembly between p35* and p40*!
!
Yield after SEC purification: 1.9 mg/L ( ~12% of yield pre SEC purification)
Yield after SEC purification: 1.3 mg/L ( ~5% of yield pre SEC purification)
5 10 15 20
0
5
10
15
20
25
p35−scFv(F8) 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
2
4
6
8
10
p35−Db(F8) S200 10−300
Volume [mL]
mAU
5 10 15 20
0
2
4
6
8
10
p40−Db(F8) S200 10−300
Volume [mL]
mAU
Yield after SEC purification: 0.6 mg/L ( ~9% of yield pre SEC purification)
-‐10
10
30
50
70
90
110
130
-‐50 50 150 250 350 450
p35-‐Db(F8) + p40-‐Db(F8)
-‐10
10
30
50
70
90
110
130
-‐50 50 150 250 350 450
p35-‐scFv(F8) + p40-‐scFv(F8)
-‐-‐-‐ 500 nM -‐-‐-‐ 250 nM -‐-‐-‐ 125 nM -‐-‐-‐ 62,5 nM -‐-‐-‐ 31,25 nM -‐-‐-‐ 0 nM
Kd= 35.1 nM
p35* mutants (scFv or Db)! p40* mutants (scFv or Db)!immobilized on chip!
IL-12* mutants!+
Coating: 1000 RU (p40*-scFv) 1600 RU (p40*-Db)!
SPR analysis
RU RU
10-6 10-5 10-4 10-3 10-2 10-1 1000.0
0.2
0.4
0.6
0.8
1.0
IL12 equivalents [µM|
A45
0nm
p35S92S/p40C197S reassembly
scIL12 (mouse)6xHis taggedscFv(F8) fusionsDb(F8) fusionsPBS
IC50 scFv(F8) fusions: 42.02 µM Db(F8) fusions: 16.59 µM
+
In vitro assembled! IL-12* mutants!
Mouse splenocytes! ELISA for IFN-‐γ!quanLficaLon
48 h!!37° C
Bioassay
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]m
AU5 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!
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!!!!! !!!
Acknowledgments!
l Prof. Dr. Dario Neri!l Dario Venetz!l The whole Neri lab!
Thanks! Danke! Grazie!
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