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!

2. 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!

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!

9. p35-­‐His
No
SEC
due
to
small
quanLty
of
protein
SEC
purificaLon

10. 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)

11. -­‐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

12. 10-6 10-5 10-4 10-3 10-2 10-1 100
0.0
0.2
0.4
0.6
0.8
1.0
IL12 equivalents [µM|
A450nm
p35S92S/p40C197S reassembly
scIL12 (mouse)
6xHis tagged
scFv(F8) fusions
Db(F8) fusions
PBS
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

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!
!

15. Acknowledgments!
l Prof. Dr. Dario Neri!
l Dario Venetz!
l The whole Neri lab!
Thanks!
Danke!
Grazie!