Defense of thesis presentation

  • Published on
    07-Jan-2017

  • View
    21

  • Download
    2

Embed Size (px)

Transcript

  • 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-!

    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 4C!

    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!

    The cleavage site is accessible by the TEV protease! 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 4C 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

    Both IL-12* subunits are sticky ! 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

    p35scFv(F8) S200 10300

    Volume [mL]

    mAU

    5 10 15 20

    0

    5

    10

    15

    20

    25

    p40scFv(F8) S200 10300

    Volume [mL]

    mAU

    5 10 15 20

    0

    2

    4

    6

    8

    10

    p35Db(F8) S200 10300

    Volume [mL]

    mAU

    5 10 15 20

    0

    2

    4

    6

    8

    10

    p40Db(F8) S200 10300

    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 [M|

    A45

    0nm

    p35S92S/p40C197S reassembly

    scIL12 (mouse)6xHis taggedscFv(F8) fusionsDb(F8) fusionsPBS

    IC50 scFv(F8) fusions: 42.02 M Db(F8) fusions: 16.59 M

    +

    In vitro assembled! IL-12* mutants!

    Mouse splenocytes! ELISA for IFN-!quanLficaLon

    48 h!!37 C

    Bioassay

  • 5 10 15 20

    0

    5

    10

    15

    20

    25

    Positive control S200 10300

    Volume [mL]

    mAU

    5 10 15 20

    0

    5

    10

    15

    20

    25

    p40scFv(F8) S200 10300

    Volume [mL]m

    AU5 10 15 20

    0

    5

    10

    15

    20

    25

    p35scFv(F8) S200 10300

    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 10300

    Volume [mL]

    mAU

    5 10 15 20

    0

    5

    10

    15

    20

    25

    p40scFv(F8) S200 10300

    Volume [mL]

    mAU

    5 10 15 20

    0

    5

    10

    15

    20

    25

    p35scFv(F8) S200 10300

    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!