Improving the therapeutic potential of human granzyme B and evaluation of granzyme M as novel effector molecules in cytolytic fusion proteins for the treatment of Serpin B9-positive cancer Von der Fakultät für Mathematik, Informatik und Naturwissenschaften der RWTH Aachen University zur Erlangung des akademischen Grades einer Doktorin der Naturwissenschaften genehmigte Dissertation vorgelegt von Diplom-Bioingenieurin Sonja Schiffer, geb. Hermes aus Neuss Berichter: Universitätsprofessor Dr. rer. nat. Rainer Fischer Universitätsprofessor Dr. rer. nat. Dr. rer. medic. Stefan Barth Tag der mündlichen Prüfung: 29. November 2013 Diese Dissertation ist auf den Internetseiten der Hochschulbibliothek online verfügbar.
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Improving the therapeutic potential of
human granzyme B and evaluation of
granzyme M as novel effector molecules in
cytolytic fusion proteins for the treatment of
Serpin B9-positive cancer
Von der Fakultät für Mathematik, Informatik und Naturwissenschaften
der RWTH Aachen University zur Erlangung des akademischen Grades einer
Doktorin der Naturwissenschaften genehmigte Dissertation
vorgelegt von
Diplom-Bioingenieurin
Sonja Schiffer, geb. Hermes
aus Neuss
Berichter: Universitätsprofessor Dr. rer. nat. Rainer Fischer Universitätsprofessor Dr. rer. nat. Dr. rer. medic. Stefan Barth
Tag der mündlichen Prüfung: 29. November 2013
Diese Dissertation ist auf den Internetseiten der Hochschulbibliothek online verfügbar.
ABBREVIATIONS ............................................................................................................................................... V
1.1 CANCER .................................................................................................................................................... 1
1.2 TARGETED CANCER THERAPY ......................................................................................................................... 2
1.3.1 Granzyme B ....................................................................................................................................... 5
1.3.2 Granzyme M ...................................................................................................................................... 7
1.4 TUMOR ESCAPE MECHANISMS TO EVADE IMMUNOSURVEILLANCE ......................................................................... 8
1.4.1 The granzyme B inhibitor PI-9 ........................................................................................................... 9
1.5 HODGKIN LYMPHOMA AND THE CD30 RECEPTOR ........................................................................................... 11
1.6 CHRONIC AND ACUTE MYELOMONOCYTIC LEUKEMIA AND THE CD64 RECEPTOR .................................................... 14
2.9 IN VIVO EXPERIMENTS ................................................................................................................................ 41
2.9.1 Mouse strains, housing and maintenance of animals ..................................................................... 41
2.9.2 Handling of mice and anesthesia .................................................................................................... 41
2.9.3 Establishment of xenograft subcutaneous tumor models ............................................................... 41
2.9.4 In vivo optical imaging by Cri-Maestro system ............................................................................... 42
2.9.5 Biological activity of Gb-Ki4(scFv) and GbR201K-Ki4(scFv) ............................................................. 42
4.1 THE THERAPEUTIC POTENTIAL OF GRANZYME B-BASED CYTOLYTIC FUSION PROTEINS .............................................. 86
4.2 IMPACT OF ENDOGENOUS PI-9 EXPRESSION IN CANCER CELLS ON THE PRO-APOPTOTIC ACTIVITY OF GRANZYME B ........ 89
4.3 GENERATION AND IDENTIFICATION OF A PI-9 INDEPENDENT GRANZYME B VARIANT .............................................. 94
4.3.1 Evaluation of in silico findings and comparison with in vitro enzymatic activity ............................ 94
4.3.2 Selection of the optimal granzyme B mutant .................................................................................. 95
4.4 EFFICACY OF THE GBR201K MUTANT IN VIVO ................................................................................................ 98
4.5 EX VIVO DETERMINATIONS ON PRIMARY LEUKEMIC CELLS ................................................................................ 101
4.5.1 CMML and AMML primary cells as target for CD64-specific fusion proteins ................................ 101
4.5.2 Specific cytotoxic efficacy of CD64-specific fusion proteins on primary CMML and AMML cells .. 103
4.5.3 The role of PI-9 and the potential of the determined cytolytic fusion proteins for personalized
medicine ..................................................................................................................................................... 105
4.6 APPLICATION OF GRANZYME M AS AN ALTERNATIVE EFFECTOR MOLECULE IN CYTOLYTIC FUSION PROTEINS ............... 106
4.6.1 Efficacy of granzyme M based CFPs .............................................................................................. 107
4.6.2 Clinical potential of granzyme M as effector molecule in cytolytic fusion proteins ...................... 109
5 OUTLOOK ............................................................................................................................................. 111
7 LITERATURE ......................................................................................................................................... 115
8 APPENDIX ................................................................................................................................................. I
8.1 DNA AND AMINO ACID SEQUENCE OF GBR201K ............................................................................................... I
8.2 SYNTHETIC DNA SEQUENCE OF GM-H22(SCFV) IN PMS VECTOR ......................................................................... I
9 INDEX OF FIGURES AND TABLES .............................................................................................................. IV
10 PUBLICATIONS ........................................................................................................................................ VI
the cell from the induction of apoptosis, thus the pro-peptide must be removed to generate an active
protease [39]. In the Golgi apparatus, a mannose-6-phosphate tag is attached which directs
granzyme B to the cytotoxic granules. Here the pro-peptide is removed by the dipeptidyl peptidase I
(DPPI) (also known as cathepsin C) [40]. Several strategies have been developed to accomplish the
correct in vitro or in vivo processing and activation of recombinant granzyme B, including the here
applied insertion of an enterokinase cleavage site (ECS) upstream of the sequence of the mature
granzyme B polypeptide, allowing activation after purification by in vitro processing. Secretory
expression of the active protein in P. Pastoris can be achieved by the direct N-terminal fusion of the
mature polypeptide to the Saccharomyces cerevisiae (S. cerevisiae) mating factor α prepro-leader
sequence allowing activation by local Kex2-protease. Secretion and functional in vivo activation by a
host cell signal peptidase has been achieved in insect cells and COS cells using the native sequence of
the granzyme B precursor protein with the propeptide deleted, or the genetic fusion of a furin
recognition motif allowing in vivo processing by endogenous P. pastoris furin-like proteases. In
addition, in vitro and in vivo activation has been achieved by either co-expression of rat DPPI in COS
cells or subsequent incubation with bovine spleen DPPI [4].
The crystal structure of granzyme B has been elucidated before by two groups [41,42]. As for all
proteases, the catalytic activity of granzyme B depends on a serine residue at the active site, one of a
triad of residues corresponding to His57, Asp102 and Ser195 in chymotrypsin. The key residue for
contact with the P11 substrate residue is Arg226 [34]. The optimal tetrapeptide recognition motif (P4-
P1) is IEPD, which is similar to that of caspase 8 and caspase 9 and thereby reflects their common
role in the activation of downstream caspases such as caspase-3 [42].
1.3.2 Granzyme M
Granzyme M is classified as ‘Met-ase’ since it cleaves specifically after amino acids with long aliphatic
side chains such as methionine, leucine, or norleucine at the P1 position1 [43]. It is predominantly
expressed within NK cells, NKT cells, γd-T cells and CD8+ effector T cells [44,45]. Recently, it was
shown that granzyme M is involved in TLR4-driven inflammation and endotoxicosis, and as such it
contributes to the inflammatory response to bacterial lipopolysaccharide by inducing serum
cytokines such as interferon (IFN) γ, IL-1α, IL-1β and TNF α [46]. However, the exact mechanism
remains unclear.
Many groups have reported the cytotoxic efficacy of granzyme M towards tumor cells [47,48].
However, the precise molecular mechanism remains controversial in the literature. It has been
1 The active sites in enzymes can be divided in subsides (S) which consist of single amino acids. The corresponding positions (P) of the substrate have the same numbering as the subsides they occupy. The positions are counted from the point of cleavage. P1 is the primary determinant of specificity.
stoichiometric complex, in which both proteins remain inactive
substrate. The interaction between
(stoichiometry of inhibition) ~ 1
intensively and during their studies they
binds less efficiently to PI-9 [86]. This mutant is used as
Figure 1: X-ray crystal structure of the non
(purple) and S195A trypsin (cyan) (PDB file 1K9O
To visualize the initial complex, mutated proteins were used. the surface of the protease. This complex can be used to superimpose the crystal structure of uncomplexed granzyme B (PDB file 1IAU) and the
1.5 HODGKIN LYMPHOMA
The above mentioned infiltrating immune cells
lymphoma (HL). 150 years ago Thomas Hodgkin already described several cases of a disea
was later associated with Hodgkin’s diseas
disease. Its hallmarks are the B
Reed-Sternberg cells (HRS cells), first published in 1900
1 % of cells in the tumor tissue, although some cases may show more than 10
majority of cells in Hodgkin lymphoma lesions are a mixed infiltrate of various types of cells of the
immune system including T cells, B cells, plasma cells, neutrophils,
to verify the potential of this enzyme in context of novel human
nd the CD30-targeting
targeting H22(scFv), mammalian secretory expression and purification.
negative HL or AML cell
in comparison to
Material and methods ________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________
19
2 Material and methods
2.1 CHEMICAL AND BIO-CHEMICAL MATERIAL
2.1.1 Equipment
The equipment and devices used in this work are listed below.
Autoclaves: Varioklav (H+P) 75S (Thermo Electron Corporation)
Mini Protean III gel chamber (BioRad Laboratories, Inc.), Protein Gel-Apparatus and supplies Mini PROTEAN II™ (BioRad), Gel Air Dryer (BioRad), Mini Trans-Blot Cell (BioRad)
Ice machine: Icematic D201 (Castel Mac)
Incubators: Thermomixer compact (Eppendorf AG), Incubator Function Line Type UT12 (Heraeus Instruments); cell culture: CD210 (Binder)
Liquid handling: Pipetman P (Gilson), Multichannel Pipettes (Eppendorf)
Microplate reader: ELISAreader ELx808 (Bio-TEK Instruments) and Epoch (Bio-TEK Instruments)
Sonication: Bandelin Sonopuls HD 2070 (Bandelin Electronic) Micro tip MS72/73
Transiluminator: Molecular Imager Gel Doc XR System (BioRad)
Waterbaths: TE31025, TE12000 (Sartorius)
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20
2.1.2 Chemicals and consumables
If not otherwise indicated, the chemicals used were purchased from the following companies in the
Material and methods ________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________
21
2.1.4 Synthetic oligonucleotides and plasmids
Synthetic oligonucleotides were purchased from MWG-Biotech (Ebersberg) in standard quality. The
DNA oligonucleotides used in this work are listed in Table 1.
Table 1: List of oligo nucleotides used for cloning and DNA sequencing.
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22
The underlying plasmids for all molecular cloning procedures were pMS for mammalian expression
[137] or pMT for bacterial expression [133]. All cloning work was adapted to the characteristics of the
chosen vectors, such as corresponding restriction sites, and performed according to standard
methods (2.2).
For expression of all recombinant granzyme B and granzyme M constructs the already published
plasmids pMS-L-EGb-H22-IV [138] and the earlier generated pMS-L-EGb-Ki4-IV and pMS-L-EGb-IV
were applied as template plasmids. The granzyme M sequence was prepared earlier during a
bachelor thesis by PCR using specific primers based on a full-length cDNA clone (IRATp970D0643D)
from the German Resource Centre for Genome Research (RZPD).
For expression of recombinant human PI-9, cDNA was derived from RNA preparations of target cell
lines by reverse transcription and specific primers (2.1.4). The derived sequence was either cloned
into the pMS vector, deleting the Igκ leader sequence for cytosolic mammalian expression
(pMS-PI9-IV) or into the pMT plasmid to express PI-9 in E. coli (pMT-L-PI9).
2.1.5 Antibodies and antibody fragments
The following antibodies and antibody conjugates were used in this work for western blot (2.4.2) or
flow cytometric (2.5) detection of recombinant or soluble proteins with the indicated dilutions:
- GAM-AP (1:5000) or GAM-PO (1:5000) or GAM-FITC (1:100): Goat-anti-mouse polyclonal
antibody directed against the Fc-part of murine IgGs, conjugated to Alkaline Phosphatase
(AP), horseradish peroxidase (PO) or Fluorescein-Isothiocyanat (FITC) (Sigma, Munich)
- anti-His (1:5000): monoclonal mouse-anti-penta-His antibody directed against C- or
N-terminal Histidin Tags (His-tags) of recombinant proteins (Sigma, Munich)
directed against the C- or N-His-tags of recombinant proteins, conjugated to fluorescent dye
Alexa Fluor 488 (Qiagen, Hilden)
- anti-human Gb-PE (1:50): monoclonal mouse-anti-granzyme B-PE antibody directed against
human granzyme B, conjugated to R-Phycoerythrin (PE) (BD Bioscience, Heidelberg)
- anti-human Gm (1:500): monoclonal mouse-anti-granzyme M antibody directed against
human granzyme M, clone 4D11 (Abnova, Heidelberg)
- anti-human Gb (1:1000): monoclonal mouse-anti-granzyme B antibody directed against
human granzyme B, clone 2C5 (Santa Cruz, San Diego)
- anti-human PI-9 (1:300): monoclonal mouse-anti-PI-9 antibody directed against human PI-9,
clone 7D8 (Santa Cruz, San Diego)
Material and methods ________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________
23
The following antibodies were used for the flow cytometric (2.5) detection of surface antigens on
cancer cell lines or primary leukemic cells according to the manufacturer’s instructions. They are
monoclonal mouse-anti-human antibodies conjugated either to FITC, PE, Allophycocyanin (APC),
Alexa 647 or 488 and detect the antigens as indicated by their name.
- anti-CD64-FITC (AbD Serotec, Duesseldorf)
- anti-CD64-488 (Biolegend, San Diego, USA)
- anti-CD64-Alexa 647 (AbD Serotec, Duesseldorf)
- anti-CD14-APC (eBioscience, Frankfurt)
- anti-CD14-FITC (Dako, Hamburg)
- anti-CD56-APC (eBioscience, Frankfurt)
- anti-CD56-PE (eBioscience, Frankfurt)
- anti-CD33-APC (eBioscience, Frankfurt)
- anti-CD25-PE (eBioscience, Frankfurt)
In addition, the single chain antibody fragments (scFv) listed in Table 2 were used in this study.
Table 2: Origin and specificity of antibody fragments.
Antibody/ligand specificity origin/specification
H22(scFv) human CD64 humanized monoclonal antibody H22
Ki4(scFv) human CD30 mouse monoclonal antibody Ki4
425(scFv) human EGF receptor, extracellular domain III (no mouse cross reactivity)
mouse monoclonal antibody 425
2.1.6 Bacterial strains
The following E. coli strains were used for selection and amplification of plasmid DNA, and for
Material and methods ________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________
24
2.1.7 Mammalian cell lines
The list in Table 3 displays all mammalian cell lines used, including their background and indicating
the receptor that played a role in this work.
Table 3: Mammalian cell lines.
Name Background Receptor
used
Reference / Source
HEK293T highly transfectable derivative of the human primary embryonal kidney cell line 293
/ ATCC, CRL-11268
HL60 established from the peripheral blood of a 35-year-old woman with AML FAB M2 in 1976
have to be stimulated with IFNγ (50 U/mL) 24 h prior to cytotoxicity/ apoptosis assays [139]
CD64 DSMZ, ACC 3
L540 established from the bone marrow of a 20-year-old woman with HL
CD30 DSMZ, ACC 72
L540cy subline of L540: was recultured from a tumor grown in a nude mouse after injection of L540 and treatment of the mouse with cyclophosphamide
CD30 [140]
L428 established from the pleural effusion of a 37-year-old woman with HL 1978
for mouse experiments transfected with far red fluorescent protein Kat2 (pTag-Katushka2-N; Evrogen)
CD30 DSMZ, ACC 197
L1236 established from the peripheral blood of a 34-year-old man with HL in 1994
CD30 DSMZ, ACC 530
K562 established from the pleural effusion of a 53-year-old woman with CML in blast crisis in 1970
CD30 DSMZ, ACC 10
Jurkat established from the peripheral blood of a 14-year-old boy with acute lymphoblastic leukemia (ALL) at first relapse in 1976
/ DSMZ, ACC 282
A431 established from the solid tumor of an 85-year-old woman (epidermoid carcinoma)
EGFR DSMZ, ACC 91
Ramos established from the ascitic fluid of a 3-year-old boy with American-type Burkitt lymphoma in 1972
/ DSMZ, ACC 603
Karpas 299 established from the peripheral blood of a 25-year-old man with T cell non-HL in 1986
/ DSMZ, ACC 31
Kasumi-1 established from the peripheral blood of a 7-year-old Japanese boy with AML FAB M2 (in 2nd relapse after bone marrow transplantation) in 1989
/ DSMZ, ACC 220
Material and methods ________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________
25
2.1.8 Primary cells
Primary cells from patients were obtained through PD Dr. med. Edgar Jost (University Hospital of
Aachen) after informed consent and with the approval of the Clinical Research Ethics Board of the
RWTH Aachen University. The patients’ diagnosis was either AMML or CMML 1 (here abbreviated as
CMML). Diagnosis of CMML was made according to the commonly used WHO-criteria. No prior
treatment occurred for any patients except for CMML II (2000 mg/d hydroxyurea) and CMML III
(1500 mg/d hydroxyurea). Mononuclear cells were isolated from peripheral blood by density
gradient centrifugation using Biocoll separating solution (Biochrom AG) and subsequently cultured in
RPMI complex medium (2.3). Prior to cytotoxicity and apoptosis assays they were stimulated with
Material and methods ________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________
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27
Their application will be described in later chapters. All media were autoclaved for
20 min/121 °C/1 bar. If necessary, buffers were sterile filtrated with a 0.2 µm PES membrane. The
following antibiotics were applied in media for bacterial cultivation, if necessary, with the indicated
The described methods followed standard techniques according to Sambrook and Russell [141].
2.2.1 Polymerase chain reaction
The polymerase chain reaction (PCR) for the amplification of DNA segments was performed using
Phusion High-Fidelity DNA Polymerase (NEB), a proofreading polymerase with 3´→5´ exonuclease
activity resulting in higher accuracy during amplification. The standard volume of a PCR procedure
was 50 μL and composed of 1x HF buffer including MgCl2, 10 pmol 5’ primer, 10 pmol 3’ primer,
0.2 mM dNTPs. After the addition of 50 ng of the template and double distilled water (ddH2O),
0.5 % (v/v) polymerase was added.
The following standard protocol with 30 cycles was used:
30 sec 98 °C Loop30 cycles start 10 sec 98 °C
20 sec 55 °C 30 sec 72 °C
Loop30 cycles end 5 min 72 °C
To verify the successful transformation of a specific insert into bacterial cells, a colony PCR reaction
was run using home-made Taq DNA polymerase. Therefore colonies were picked and heated at 95 °C
for 15 min in 20 µl ddH2O in order to destroy the bacterial cells. After spinning off the cell debris
10 µl of the mixture was added to the PCR mixture containing 1x PCR buffer, 1 mM MgCl2, 10 pmol
5’ primer, 10 pmol 3’ primer, 0.2 mM dNTPs, 1 U Taq DNA polymerase in 25 µL total volume.
The following protocol with 30 cycles was applied:
5 min 96 °C Loop30 cycles start 1 min 96 °C
30 sec 55 °C 45 sec 72 °C
Loop30 cycles end 5 min 72 °C
Material and methods ________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________
28
The purification of the PCR products was done according to the manufacturer’s instructions
(Macherey-Nagel Kits, 2.1.3).
2.2.2 Site-directed mutagenesis
Site-directed mutagenesis was conducted by overlap extension PCR using specific primers during an
SOE (splicing by overlapping extension) PCR. The primer pairs for PCR 1 containing the corresponding
mutations are listed in Table 1. To obtain the two fragments, the primer 5’ pMS was used to generate
the 5’ to 3’ fragment and the primer 3’ Gb (BlpI) was used to generate the 3’ to 5’ fragment. A
construct containing the desired single chain sequence as ligand, including the wild-type sequence of
granzyme B, was used as a template. (2.1.4). The PCR mixtures and programs were as follows.
PCR 1:
1x HF buffer
10 ng template DNA
10 pmol 5’ primer
10 pmol 3’ primer
0.2 mM dNTPs
0.02 U/µL Phusion DNA polymerase
Ad 50 µL ddH2O
Program 1:
30 sec 98 °C
Loop30 cycles start 10 sec 98 °C
20 sec 60 °C
30 sec 72 °C
Loop end
5 min 72 °C
Annealing:
2 µL fragment 1
2 µL fragment 2
1x HF buffer
0.2 mM dNTPs
0.02 U/µL Phusion DNA polymerase
Ad 50 µL ddH2O
Program 2:
30 sec 98 °C
Loop7 cycles start 10 sec 98 °C
1 min 50 °C
30 sec 72 °C
Loop end
5 min 72 °C
Amplification:
10 µL annealing product
1x HF buffer
10 pmol 5’ primer (5’ pMS)
10 pmol 3’ primer (3’ Gb (BlpI))
0.2 mM dNTPs
0.02 U/µL Phusion DNA polymerase
Ad 50 µL ddH2O
Program 3:
30 sec 98 °C
Loop25 cycles start 10 sec 98 °C
20 sec 60 °C
30 sec 72 °C
Loop end
5 min 72 °C
Material and methods ________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________
29
2.2.3 Agarose gel electrophoresis
To analyze the size and concentration of the PCR products (2.2.1), isolated plasmids (2.2.8) and
restriction fragments (2.2.5), analytical agarose gel electrophoresis was carried out in 1.2 % (w/v)
agarose at field strength of 120 V after addition of 5x OrangeG loading buffer (Table 4).
Electrophoresis was run in 1x TAE buffer with 0.3 μg/mL ethidium bromide. 0.33-0.6 μg 2-Log DNA
Ladder (NEB) was used as the standard for determining the size and concentration of the fragments
(2.1.3). DNA bands were visualized on a UV transilluminator at a wavelength of 302 nm. The bands
obtained were documented with a video documentation system using the software ‘Quantity One
Document Software’ version 4.6 (BioRad, Munich).
2.2.4 Zero blunt TOPO PCR cloning
Since the digestion of PCR products (2.2.1) was often inefficient, the PCR products were cloned into a
TOPO vector according to the manufacturer’s instructions of Zero Blunt® TOPO® PCR Cloning Kit
(Invitrogen, Darmstadt). After growing of the corresponding bacteria, the purified plasmids could be
digested as described below (2.2.5).
2.2.5 Endonuclease restriction of DNA
The double digestion with endonucleases and buffers from NEB (Frankfurt am Main) was performed
according to the manufacturer’s instructions in a total volume of 50 µL with 1 to 2 µg DNA. The
isolation of restricted DNA occurred via preparative agarose gel electrophoresis (2.2.3). After
separation of DNA fragments, desired DNA bands were excised using a clean scalpel on a UV
transilluminator. The isolation of the DNA from the gel was performed according to the
manufacturer's protocol (2.1.3). The concentration of the purified DNA was determined using
analytical agarose gel electrophoresis (2.2.3) or by measuring the absorbance at 260 nm. The
extracted and purified DNA was then used for ligation (2.2.6). For control digestions, 5 times less
than the recommended volume was used and the specific bands were visualized as described above
(2.2.3).
2.2.6 Ligation of DNA fragments
Two purified restriction digested fragments (2.2.5) were ligated with T4 DNA ligase and Quick ligase
respectively, using the buffer system specified by the manufacturer with a total volume of 20 μL. The
ligation mix contained the insert in triplicate molar excess of vector quantity, and the mixture was
incubated overnight at 16 °C and for 5 min at room temperature (RT) respectively. Afterwards, the
whole ligation mixture was transformed into heat shock competent bacteria (2.2.7), as explained
Material and methods ________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________
30
below.
2.2.7 Transformation of plasmid DNA into competent E. coli
Heat shock competent cells (2.1.6) were prepared with Rhubidium-chloride according to Hanahan
[142]. The aliquoted cells were thawed on ice before the DNA was added (using either 20 µL ligation
mix (2.2.6) or 30 ng purified plasmid DNA (2.2.8) for re-transformation). After incubation for 30 min
on ice, the cells were incubated at 42 °C for 30 sec (heat shock). The transformed bacteria were
stored on ice for 2 min and then mixed with 800 μL of sterile SOC medium. For regeneration, the
bacteria were incubated for 1 h at 37 °C before they were plated on LB agar plates (2.1.9) containing
the corresponding antibiotic (ampicillin for pMS-plasmids, kanamycin for pMT-plasmids). The plates
were incubated overnight at 37 °C and colonies were analyzed using colony PCR (2.2.1) the next day.
Replica plates were prepared for at least three clones.
For the final clones glycerol stocks were prepared in 25 % (v/v) glycerol solution and stored at -80 °C.
2.2.8 Preparation of plasmid DNA from E. coli
For small scale DNA preparation, 3 mL of LB medium supplemented with the appropriate antibiotic
(2.1.9) was inoculated and then the recombinant plasmid DNA (2.2.8) was isolated using a
NucleoSpin plasmid kit according to the manufacturer’s instructions (2.1.3). The concentration was
determined at 260 nm.
2.2.9 Sequencing of DNA
To verify the desired sequence of recombinant clones (2.2.7, 2.2.8), sequencing was carried out at
the Fraunhofer IME sequencing facility by the chain termination method using the ‘Applied
Biosystems 3700 DNA Analyzer’ and the BigDye™ cycle sequencing kit. 160 ng/kb plasmid DNA was
added to ddH2O in a total volume of 28 μL with 20 pmol of sequencing primer (2.1.4). The resulting
sequences were evaluated with the ‘CLC Main Workbench 6.0’ and ‘Clone Manager 9.0’ software.
2.3 CELL CULTURE
2.3.1 Cultivation of cell lines and primary cells
All mammalian cell lines (Table 3) as well as the primary monocytic cells (2.1.8) were cultured in T25
or T75 cell culture flasks (Greiner) in RPMI1640 (Gibco) supplemented with 10 % (v/v) heat-
inactivated fetal calf serum (FCS) (Gibco), 50 μg/mL penicillin and 50 μg/mL streptomycin. The cells
Material and methods ________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________
31
were maintained at 37 °C in a humidified atmosphere of 5 % CO2. After transfection of HEK293T cells,
100 µg/mL Zeocin was added for selection purposes.
When large amounts of secretory expressed protein were intended, triple flasks (VWR, Darmstadt) or
cell factories (Thermo Fisher Scientific, Waltham, USA) were inoculated, so supernatant volumes
above 1 L could be collected easily for purification.
For viability and apoptosis assays 200 or 50 U/mL Interferon (IFN) γ was added for stimulation of
CD64 expression in the primary cells or HL60 cell line, respectively.
2.3.2 Transfection of cell lines
The physical or chemical transfection of different mammalian cell lines was performed according to
the manufacturer’s instructions (Table 5) and using their protocols for optimization. As
recommended, the cells were kept in serum-free RPMI medium or RPMI containing 10 % (v/v) FCS
without antibiotics. The efficiency of transfection was monitored via flow cytometric analysis.
Table 5: Reagents for physical or chemical transfection of mammalian cell lines.
Name Company
Physical:
Amaxa Nucleofection Kit V Lonza (Cologne)
Neon Transfection systems Life technologies (Darmstadt)
Chemical:
Nanofectin Kit PAA (Pasching, Austria)
Attractene Transfection reagent
Qiagen (Hilden)
GeneJuice Transfection Reagent
Novagen (Darmstadt)
TransIT-Jurkat MirusBio (Madison, USA)
Ingenio Electroporation Kit MIR 50109
MirusBio (Madison, USA)
Roti-Fect Reagent Roth (Karlsruhe)
2.3.3 Protein delivery into cells via streptolysin O
Reversible permeabilization of cells can be achieved via streptolysin O (Sigma, Munich). For protein
delivery 5*106 cells/mL were washed once with HEPES buffer (20 mM HEPES, pH 7.4, 150 mM NaCl)
and re-suspended in 100 µL HEPES buffer containing 0.5 % (w/v) BSA, 5 mM DTT and 10 mM glucose.
Material and methods ________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________
32
The cells were incubated with 8 µg/mL streptolysin O for 15 min at 37 °C in a 96-well plate.
Afterwards the cell-death inducing protein was added and cells were further incubated for 4 h. The
permeabilization step was conducted in the absence of Ca2+, whereby resealing of the cells was
induced by addition of 1.25 mM Ca2+ 1 h after addition of the cell-death inducing protein. Apoptotic
effects were monitored via Annexin V/ PI assay as described in 2.7.2.1.
2.4 PROTEIN-BIOCHEMICAL METHODS
2.4.1 SDS-PAGE
To determine the amount and identity of proteins (2.4.3, 2.4.4) according to their molecular weight,
analytical protein separation was performed via discontinuous SDS- poly-acryl-amide (SDS-PAA) gel
electrophoresis (SDS-PAGE) based on the method of Laemmli [143]. The denaturing SDS-PAA gel was
prepared with the above mentioned buffers (Table 4). Before the gel was loaded, the samples were
mixed with Laemmli sample buffer and heated for 5 min at 95 °C to destroy the structure of the
proteins. The gel was run with 160 mV in ‘Mini-Protean III’-protein gel electrophoresis chamber
(BioRad) until the samples reached the bottom of the gel. The gel was dyed in a coomassie brilliant
blue solution for 10-30 min and destained with the described buffer (Table 4). With the help of the
loaded standard (2.1.3) the proteins could be identified.
2.4.2 Western blot analysis
Western blot analysis was performed using the ‘Mini Trans-Blot® Electrophoretic Transfer Cell’ (BIO-
RAD) according to the manufacturer's instructions with transfer buffer (Table 4). After the transfer
(250 mA for 90 min or 40 V overnight at 4 °C) of proteins, separated by SDS-PAGE, on a nitrocellulose
membrane (Protran, Whatman), blocking was performed using 2.5 % (w/v) milk powder resolved in
PBST either for 1 h at RT or overnight at 4 °C. After washing three times with PBST, the membrane
was incubated with the primary antibody in PBST for 1 h at RT. The same procedure was followed for
incubation with the secondary antibody. After final washing in PBST and water, the membrane was
developed in the dark depending on the conjugation of the second antibody (2.1.5) if not otherwise
indicated using NBT/BCIP [3.33 % (w/v) NBT, 1.67 % (w/v) BCIP in dimethylformamide] added to the
100x volume of AP buffer (Table 4) for AP-labeled antibodies or with an enhanced
chemiluminescence (ECL) system (Thermo Scientific) and LAS-3000 reader (Fujifilm) for PO-
conjugated ones.
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33
2.4.3 Bacterial expression and purification
For the expression of recombinant PI-9, the E. coli expression strain BL21 (DE3) was used (2.1.6). To
evaluate successful protein expression, optimal conditions and sufficient purification steps, small
scale experiments were performed in a volume of 300 mL according to the published stress-
expression protocols [144] or the protocols from Qiagen (‘The QiaExpressionistTM’). In order to obtain
enough protein for further determinations, fermentation with 4 L volume (BioFlo 110 fermenter,
400-1000 rpm) was run in synthetic minimal medium (16.6 g/L KH2PO4, 4.6 g/L NH4(H2PO4), 70 mg/L
supplemented with 20 g/L glucose. Inoculation was prepared with 400 mL bacteria culture grown at
37°C overnight in LB medium. The bacteria were harvested 24 h after induction with 1 mM Isopropyl
Thiogalactoside (IPTG).
After harvesting and centrifugation (4000 g, 15 minutes, 4°C) the bacterial pellet was re-suspended in
lysis buffer (50 mM NaH2PO4, pH 8.0, 500 mM NaCl, 0.5 mM DTT) and sonicated six times for 60 s,
70 %, 9 cycles (2.1.1). Due to its enzymatic activity towards proteases, the addition of protease
inhibitor was disclaimed. Cell debris was removed by centrifugation at 30.000 g, 4 °C for 30 min. The
supernatant was supplemented with 10 mM imidazol and purified via Immobilized Metal-ion Affinity
Chromatography (IMAC) and Fast Performance Liquid Chromatography (FPLC) as described below
(2.4.4.2). After a second IMAC step, the protein was re-buffered into 20 mM Tris, pH 7.4, 1 mM DTT
and further purified via anion exchange chromatography (Q-Sepharose XL, 1 mL, GE Healthcare) with
a salt gradient of 0.05–1 M NaCl. To achieve satisfying purity, gel filtration chromatography followed
using a Superdex 75 (GE Healthcare) column in 20 mM Tris, pH 7.5, 50 mM NaCl and 1 mM DTT.
2.4.4 Mammalian expression and purification
All granzyme B- and granzyme M-based constructs were produced secretory in mammalian HEK293T
cells. In addition, PI-9 was expressed cytosolically within these cells.
2.4.4.1 Expression of recombinant protein in HEK293T cells
HEK293T cells were used as the expression cell line (2.1.6). The cells were transfected with 1 µg DNA
according to the manufacturer’s instructions using RotiFect (Roth) (2.3.2). The pMS plasmid used
(2.1.4) encodes for the bicistronic EGFP reporter, so expression of the target protein could be verified
by the green fluorescence via fluorescence microscopy. To obtain high producing cultures
fluorescence activated cell sorting was performed to select high reporter EGFP producing cells.
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34
The secretory expression was enabled by the signal peptide sequence of immunoglobulin kappa light
chain (Igκ) encoded by the pMS plasmid (2.1.4). Protein containing supernatants were kept sterile
and were stored at 4 °C until sufficient volumes were collected for purification (2.4.4.2). In case of
cytosolically expressed protein, the cells were harvested, washed with PBS and stored at -20 °C until
further processing (2.4.4.3).
2.4.4.2 Purification of recombinant protein from supernatant
The secreted protein could be purified from the cell culture supernatant via IMAC and FPLC. The
cleared supernatant was supplemented with 10 mM imidazole before loading onto an XK16/20
column (Amersham/ GE Healthcare) containing 8 mL Sepharose 6 Fast Flow resin (GE Healthcare).
The buffers used are listed in Table 4. Afterwards the elution buffer was exchanged with adequate
buffer to enable enterokinase digestion (20 mM Tris-HCl, 200 mM NaCl) via a HiPrep 26/10 desalting
column (GE Healthcare). To obtain higher purity of proteins especially for in vivo experiments, the
protein solution was additionally loaded onto a gel filtration chromatography column (Superdex 75,
GE Healthcare) using the enterokinase buffer.
In order to concentrate the protein solutions, Vivaspin 6 ultrafiltration spin columns (10 kDa MWCO;
Sartorius) were centrifuged at 4000 g and 4 °C. Aliquoted proteins were stored at -80 °C. To activate
granzyme-based constructs enterokinase was added to the protein (0.02 U/µg) with 2 mM CaCl2 for
16 h incubation at 23 °C prior to use. Activated protein was stored after the addition of 1 mM DTT at
-80 °C. The purified proteins or cell lysates were analyzed via SDS-PAGE under reducing conditions
and coomassie staining (2.4.1) or western blots (2.4.2).
2.4.4.3 Purification of recombinant protein from cell lysate
HEK293T cells transfected with pMS-PI9-IV (2.1.4) were harvested and lysed within a native buffer
(50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 1 % (v/v) Triton X100). Incubation of the cells in buffer took
place for 30 min on ice. Cell debris was then removed by centrifugation at 13.000 g for 15 min at 4 °C
and the supernatant containing the desired protein was purified as described in 2.4.4.2.
2.4.5 Protein quantification
Three different methods were applied for the determination of protein concentration. For purified
protein the concentration was determined after SDS-PAGE and coomassie staining using ‘AIDA Image
Analyzer’ Software (Raytest Isotopenmessgerate). The concentration could be correlated to the
additionally loaded bovine serum albumin (BSA) standard (100 ng, 250 ng, 500 ng, 1000 ng). The
values were confirmed by Bradford assay in a 96-well plate using 1x Roti-Nanoquant (Roth). All
samples were pipetted in triplicates. A stock solution with 1 μg/μL purified BSA (0, 0.5, 1, 2, 4, 6, 8,
Material and methods ________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________
35
10 μg/well) was used as a calibration standard. In order to determine the total protein concentration
of cell extracts Bradford or BCA assay (Thermo Scientific Pierce) was performed according to each
manufacturer’s instructions. Absorption was measured at 595 nm for BSA and at 562 nm for BCA in a
microplate reader. Data were analyzed with MS Office Excel software, and the total protein
concentration of the samples was determined according to the BSA standard curve.
2.4.6 Labeling of SNAP constructs
The establishment of the SNAP (engineered version of the human DNA-repair enzyme O(6)-
alkylguanine DNA alkyltransferase) -tag technology and the preparation of the corresponding
constructs and their covalent conjugation to substrates containing O(6)-benzylguanine (BG) via a
stable thioether bond in a rapid and highly specific self-labeling reaction has been described before
[145]. Here, in HEK293T cells, secretory expressed and subsequently purified SNAP-tag fusion
proteins (Ki4(scFv)-SNAP and H22(scFv)-SNAP) were conjugated with the BG-modified dyes BG Alexa
Fluor 647 (BG 647), BG Alexa Fluor 747 (BG 747) or BG Vista Green (Covalys Biosciences AG,
Witterswil) by incubation in the dark with 1.5-3-fold molar excess of the dye overnight at 4 °C.
Residual dye was removed using PD 10 columns (GE Healthcare). Stained proteins were visualized
after separation by SDS-PAGE with either a UV transilluminator (BioRad Gel Doc XR) or with a CRi
Maestro imaging system using blue, red and near-infrared filter sets.
2.4.7 Detection of endogenous PI-9 from cell lines or primary cells
For the detection of cytosolic expressed PI-9 within tumor cell lines or primary cells, 106 cells were
lysed within 50 µL lysis buffer (PBS supplemented with 1 % (v/v) Triton X-100) for 30 min on ice. The
cell extract was cleared via centrifugation and the protein concentration was determined with
Bradford reagent (2.4.5). 40 µg total protein amount were loaded on a SDS-gel for western blot
analysis. PI-9 was detected by incubation for 1 h with anti-human PI-9 (2.1.5) in PBST. After washing
with PBST, GAM-PO was added and signals could be detected as described in 2.4.2.
In parallel, detection was performed via flow cytometry (2.5.1) which allows more quantitative
analysis. Therefore 106 cells were washed with PBS and resuspended in 500 µl Cytofix/ Cytoperm (BD
Bioscience, Heidelberg). After incubation for 20 min on ice and washing of the cells, a blocking step
was included with 5 % (w/v) BSA in 200 µl PBS for 20 min on ice. The cells were washed with PBS
containing 0.2 % (v/v) Tween 20 and incubated with anti-human PI-9 (2.1.5) for 30 min on ice. The
second antibody GAM-FITC (2.1.5) was added to the washed cells and incubated as described above.
The final wash step and resuspension of the cells was performed in PBS comprising 0.2 % (v/v)
Tween 20. The measured shift during flow cytometric analysis due to a specific binding of the
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36
antibody to the fixed and permeabilized cells was quantified as MFI (Median Fluorescence Intensity)
in WinMDI 2.8. MFIs due to low unspecific binding of the secondary antibody were subtracted.
2.4.8 Serum stability assays
70 ng/µL purified protein was incubated with 50 % murine serum (AbD Serotec, Duesseldorf) or
human serum (obtained by centrifugation of 500 to 1000 µL patient-derived blood samples (2.1.8)
for 10 min, 300x g at RT) at 37 °C for different time periods between 0 and 48 h. The amount of
functional remaining protein binding was measured via flow cytometric analysis (2.5.1), here with
400-fold dilution of 7.5 µL of the mixture.
2.4.9 ELISA for detection of CD30 receptor in blood
Mouse serum of L540cy bearing mice was obtained by centrifugation of 10-30 µL mouse-derived
blood samples for 10 min and 300x g at RT and stored at -20 °C until use. The concentration of
soluble CD30 receptor within blood serum was evaluated by an ELISA set-up as previously published
[146]. High binding ELISA plates were coated with 50 µl Ki2 monoclonal antibody (240 µg/mL) in PBS
overnight at 4 °C. After washing with PBST and blocking in PBS containing 5 % (w/v) milk powder for
1 h at RT, 50 µl CD30-containing mouse serum (1:5 dilution in PBS) was added and incubated for 2 h
at RT. Plates were washed three times and 50 µl Biotin coupled Ki3 monoclonal antibody (270 ng/µL,
1:100 dilution in PBS) was added for a 1 h incubation at RT. For detection, plates were first incubated
with 50 µl Streptavidin-PO (1:200 in PBS, Abcam, Cambridge, UK) for 1 h; then washed in PBST and
then incubated for 1 h with 50 µl ABTS solution (Roche Diagnostics, Mannheim). Plates were read out
at 405 nm in a microplate reader.
2.5 FLOW CYTOMETRIC ANALYSIS
2.5.1 Binding analysis
The binding of fusion proteins to the target cells (Table 3) or the endogenous protein thereof (2.4.7)
was evaluated via flow cytometric analysis (CellQuest Version 3.3 (Becton Dickinson, Heidelberg))
and WinMDI 2.8. For standard binding analysis 4*105 cells were washed with PBS and incubated with
50 nM purified protein in 100-300 µL PBS for 30 min on ice. After 2 wash cycles the cells were
incubated with anti-His Alexa 488 (2.1.5) for 30 min on ice, in the dark. Negative controls with cell
lines not expressing the target receptor were run in parallel. Monocytic cells of primary PBMCs were
gated in forward and sideward scatter (FSC/ SSC) dotplot prior to evaluation.
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37
2.5.2 Determination of Kd values
For the determination of the affinity constant (Kd) different concentrations of the fusion proteins
(0-16.7 nM, 1:2 dilution) were incubated with the cells and evaluated by flow cytometric analysis
(2.5.1) and non-linear-regression determinations in GraphPad Prism 4.0.
2.5.3 Determination of specific cell death of primary cells
The specific reduction of the target cell population was determined by staining primary cells with
anti-CD64 Alexa647 (AbD Serotec, Duesseldorf) to localize the population within the FSC/SSC dotplot
during flow cytometry. These cells were gated with WinMDI 2.8 so their reduction after treatment
could be evaluated accordingly and compared to untreated control.
To further specify reduction of the target cell population specific antibodies displayed in 2.1.5 were
incubated with the treated cells and reduction of the stained target cell population could be
compared to the untreated control by flow cytometric analysis.
2.6 CONFOCAL MICROSCOPY
All images were taken with a LEICA confocal microscope (TCS SP5) equipped with the following
lasers: 458 nm, 488 nm, 561 nm and 633 nm. A 40x optical magnification was used. Cells were
stained with SNAP-based constructs (2.4.6) as indicated and prepared as described above for flow
cytometry (2.5), resuspended in 50 µL PBS and pipetted onto a microscope slide.
2.7 FUNCTIONAL CHARACTERIZATION OF RECOMBINANT PROTEINS
2.7.1 Protease assays
2.7.1.1 Colorimetric substrate assays
The activity of 100 nM granzyme B-based constructs after enterokinase digestion was detected by
cleavage of 200 µM of the synthetic substrate Ac-IETD-pNA (Calbiochem/Merck, Darmstadt) which
mimics the cleavage site of pro-caspase 3. The reaction was monitored in 96-well plates in a
microplate reader at 405 nm and 37 °C at 1 or 2 min intervals for 1 h. Afterwards, time was plotted
against the absorbance so differences in activity could be evaluated. After complex formation
(2.7.1.4) and measurement of the remaining activity the velocity of the reaction was calculated from
the linear slope of the reaction within the first 10-12 min and converted to pmol/min with the help of
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38
the corresponding conversion factor (µM/A405nm) according to the manufacturer’s protocol of the
Caspase-8 Assay kit (Calbiochem).
Granzyme M activity, after enterokinase digestion, was evaluated using the colorimetric substrate
Suc-Ala-Ala-Pro-Leu-pNA (Bachem, Bubendorf, Switzerland) according to a published protocol [56].
The change in absorbance at 405 nm was measured as above with the difference that here only the
absolute change was recorded, not the kinetics.
2.7.1.2 Determination of Michaelis-Menten Kinetics
To analyze the activity of the wild-type protein and its most promising mutant, Km values were
evaluated according to the manufacturer’s protocol of the Caspase-8 Assay kit (Calbiochem). The
enzymatic reaction was prepared and measured as described above (2.7.1.1). For evaluation, change
in absorbance at 405 nm during the first 12 min of the reaction was determined. Km values were
derived by non-linear curve fitting to the Michaelis–Menten equation using GraphPad Prism 4.0.
2.7.1.3 Cleavage of PI-9 by granzyme M
The activity of the granzyme M-based constructs was verified by their ability to cleave recombinant
PI-9 (1.3.2). The fusion protein was incubated at a 1:3 molar ratio with PI-9 at 25 °C in 100 mM HEPES
(pH 7.4), 200 mM NaCl, 0.01 % (v/v) Tween 20 and 1 mM DTT. After 24 h the reaction mixture was
analyzed by SDS-PAGE and western blotting using anti-human PI-9 and GAM-PO as described in 2.4.2.
2.7.1.4 Formation of the granzyme B and PI-9 Complex
The complex of recombinant PI-9 and Gb-H22(scFv) and its mutants was formed using a 5:1 molar
ratio (PI-9:Gb-H22(scFv)) under reducing conditions in 20 mM Tris-HCl (pH 7.4), 50 mM NaCl and
1 mM DTT. 600 ng of wild-type or mutant granzyme B construct was incubated with or without PI-9
for 1 h at 37 °C and the retained enzymatic activity was evaluated as described above (2.7.1.1). For
western blot analysis (2.4.2) adequate amounts containing at least 100 ng of granzyme B-based
protein was prepared for SDS-PAGE in the same manner. For detection of specific bands, anti-human
PI-9, anti-human Gb or anti-His were used followed by the second antibody GAM-PO (2.1.5).
Endogenous complex formation was determined after incubation using Gb-Ki4(scFv) as described for
apoptosis assay (2.7.2.1). Analysis was done as described above using anti-human Gb and GAM-PO.
Material and methods ________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________
39
2.7.2 Apoptosis assays
Apoptosis was documented via Annexin V/ propidium iodide (PI) staining (2.7.2.1) or via
determination of caspase 3/7 activation (2.7.2.2) within the target cells.
2.7.2.1 Annexin V/ PI Assay
Annexin V binds to phosphatidylserine which is exposed on the surface of apoptotic cells. Thus, dead
cells can be visualized using labeled Annexin V protein. In addition, PI enters through the permeable
membrane of dead cells, so necrotic and apoptotic cells can be distinguished. 2*105 cells/mL were
incubated at 37 °C, 5 % CO2, with different concentrations of fusion protein (between 11 and
100 nM), depending on target cells and construct, for 48 h in 12-well plates containing 1 mL
supplemented RPMI medium (2.3.1). Afterwards, the cells were washed in PBS and the pellet was re-
suspended either in 100 µL 1x Annexin V binding buffer (10 mM HEPES (pH 7.5), 140 mM NaCl,
0.5 mM CaCl2) supplemented with 2.5 µL Annexin V-FITC (eBioscience, Frankfurt) or in 450 µL cell-
free culture supernatant from HEK293T cells expressing Annexin V-labeled green fluorescent protein
(EGFP, [147]) supplemented with 10x Annexin V binding buffer and 5 µg/mL PI. The incubation with
labeled Annexin V protein took place in the dark, either at RT for 15 min or on ice for 20 min
respectively. Results were analyzed via flow cytometry (2.5).
2.7.2.2 Caspase 3/7 assay
To determine the caspase 3/7 activity within apoptotic, granzyme B-treated cells (2.7.2.1), a pre-
luminescent caspase 3/7-DEVD-aminoluciferine substrate was applied (Caspase-Glo™ 3/7 Assay,
Promega). Caspase 3/7 is a direct substrate of granzyme B. If it is cleaved after granzyme B delivery
into the target cells and thereby activated, it can cleave the pre-luminescent caspase 3/7-DEVD-
aminoluciferine substrate so free unbound aminoluciferine is released. This is subsequently used by
luciferase whereby a luminescence signal is produced. For the evaluation, cells were transferred into
96-well plates and the read out was done in a microplate reader. The amount of luminescence was
directly proportional to the activity of caspase 3/7.
2.7.3 Viability assay
The cytotoxic effect of the granzyme B-based fusion proteins was monitored using the ability of
metabolic active cells to reduce the tetrazolium salt XTT ((sodium 2,3,-bis(2-methoxy-4-nitro-5-
sulfophenyl)-5-[(phenylamino)-carbonyl]-2H-tetrazolium) inner salt, Life Technologies (Invitrogen)) to
orange colored compounds of formazan. The dye intensity was measured by a microplate reader and
is directly proportional to the number of living cells.
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40
For Gb-Ki4(scFv) and its mutants 2*105 cells were plated in 1 mL in 12-well plates with 1:3 dilutions
and incubated for 48 or 72 h at 37 °C and 5 % CO2. For Gb-H22(scFv) and its variants and
Gm-H22(scFv), cells were incubated in 96-well plates with 5.000 IFNγ stimulated HL60 cells (or
control cell lines) or 20.000 primary cells per 100 µL with 1:3 or 1:5 dilutions of protein for 72 h at
37 °C. In a different approach, XTT was used for read out of viable cells after incubation with single
doses of fusion protein (concentrations as indicated, depending on target cells and construct) in 12-
well plates and evaluated after transfer of 100 µL of each approach into 96-well plates as described
above. 50 µL XTT was added to a 100 µL cell suspension and extinction was measured after a 4 h
incubation at 450 nm at a reference wavelength of 630 nm.
Viability curves and IC50 values were evaluated with GraphPad Prism4.0. Therefore dilution effects by
the protein volume used were normed using respective buffer controls. Zeocin was used as the
negative control.
Competition assays were carried out with H22(scFv) by adding 10 nM Gb-H22(scFv) or Gm-H22(scFv)
to IFNγ stimulated HL60 cells and incubating them for 48 h in the presence or absence of 10 or
100 nM H22(scFv). The buffer control was assigned a viability of 100 % and the converted XTT signal
was read out as above.
2.8 IMMUNOHISTOCHEMISTRY
Resected cryo-conserved tumors were cut into 8 µm sections on a freezing microtome (Cryostat CM
3050 (Leica) and mounted on coated slides. After drying for 48-72 h, sections were fixed for 10 min
with dry acetone and air-dried. In order to detect endothelial cells, slides were incubated with
naphthol AS-BI phosphate (sodium salt, 50 mg/100 mL; Sigma, Munich) as substrate and new fuchsin
(10 mg/100 mL; Merck, Darmstadt) as chromogen dissolved in 0.1 M Tris-HCl (pH 8.5) resulting in
pink/red staining. By omitting the addition of levamisole to the reaction mixture, endothelial cells
became visible. Slides were counterstained with hematoxylin.
In order to visualize remaining CD30 receptor expression in resected tumors, 12.5 nM
Ki4(scFv)-SNAP-BG-Vista-Green diluted in PBS was added to the slides and incubated for 30 min in
the dark at RT. After washing, images were collected via a Leica confocal microscope (2.6).
Material and methods ________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________
41
2.9 IN VIVO EXPERIMENTS
The experiments were officially approved by the local Animal Care and Use Review Committee (AZ:
87-51.04.2010.A278). All animals received human care in accordance with the requirements of the
‘German Tierschutzgesetz’, §8 Abs. 1 and in accordance with the Guide for the Care and Use of
Laboratory Animals published by the National Institute of Health.
2.9.1 Mouse strains, housing and maintenance of animals
For animal experiments 6 to 8 week-old female BALBc nu/nu mice (Charles River GmbH) were used.
Mice were housed in the ‘Institut fuer Versuchstierkunde’ of the university hospital, Aachen, during
tumor take rate studies, and in the animal facility of the Fraunhofer IME during the treatment
experiments.
2.9.2 Handling of mice and anesthesia
Anesthesia of the mice was performed either by intramuscular administration of 75 mg/kg Ketamin –
10 mg/kg Xylazin for periods up to 30 min or by isoflurane for shorter times up to 2 min.
Proteins were administered intravenously (i. v.) with volumes up to 100 μL. Samples were brought up
to RT before injection. Before and after image acquisition, anesthetized mice were kept warm on a
hot-water bag. As in previous imaging experiments, the mice injected with far red fluorescent protein
Kat2 (pTag-Katushka2-N; Evrogen) transfected cells were fed a purified, chlorophyll-free diet
(AIN93G, SSNIFF GmbH) 7 days before the imaging experiments were started whereas mice injected
with L540cy were fed a normal diet (SSNIFF GmbH).
Blood samples (10-30 µL) were obtained through the tail vein of the mice.
2.9.3 Establishment of xenograft subcutaneous tumor models
Before treatment of the mice with fusion proteins could be started, the tumor take rates of the
potential target cell lines had to be determined. For injection of tumor cells, L428 cells transfected
with Kat2 or L540cy cells (not transfected) were used after washing with PBS and resuspension in
Bioscience, Heidelberg). 5*106 cells in a volume of 30 µL were injected subcutaneously into the right
hind limb of 5 mice for L428 and 5 mice for L540cy. Tumor growth was monitored by molecular
imaging with the CRi-Maestro Imaging System (2.9.4) in the case of Kat2 transfected L428; for L540cy
triplet caliper measurements were executed. Based on the evaluated tumor take rates and tumor
sizes, adequate treatment schedules could be set up.
Material and methods ________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________
42
2.9.4 In vivo optical imaging by Cri-Maestro system
In vivo optical imaging was performed with the CRi-Maestro Imaging System (Cri Inc., Woburn, MA,
USA) and analyzed with the Maestro Spectral Imaging Software. The Maestro imager has a Xenon
light source and allows measurements through the complete visible and near infrared light spectrum
by applying different filter sets. Whole mouse images were taken with constant stage height and
illumination settings. The Kat2 signal of the transfected L428 cells was visualized with the yellow filter
set (630–850 nm). To identify and discriminate the individual spectra within the images, spectral
libraries were defined. Therefore the different dye spectra were unmixed from the cube images. The
component images, which display the targeted signal, were used to define the tumor to background
ratios (TBRs) by drawing a region of interest (ROI) around the tumor and a larger area on the back
representing average background signal. The thereby calculated values for the tumor size in mm² for
the region of interest were used to calculate the tumor regression.
2.9.5 Biological activity of Gb-Ki4(scFv) and GbR201K-Ki4(scFv)
To determine the in vivo biological activity of Gb-Ki4(scFv) and GbR201K-Ki4(scFv), the cell lines L428
(Kat2 transfected) and L540cy were injected into the mice respectively as described in 2.9.3. The
mice were randomized equally into groups and received 50 µg GbR201K-H22(scFv) (as non-binding
control), Gb-Ki4(scFv) or GbR201K-Ki4(scFv) i. v. per treatment day. Tumor size was measured as
described in 2.9.3 and 2.9.4. Relative tumor size was calculated by setting the tumor size on first day
of treatment to 100 % and adapting the following values accordingly.
In vivo targeting of CD30-positive L540cy cells was accomplished by injection of 0.75 nmol
Ki4(scFv)-SNAP-BG-747 into L540cy bearing mice. 6 h later, the mice were imaged with the CRi-
Maestro system using the deep red filter set (730-950 nm) (2.9.4).
2.10 STATISTICAL ANALYSIS
Data were analyzed using a two-tailed t-test in GraphPad Prism 4.0, with p < 0.05 considered as
inhibitor. The latter was recultured from a tumor grown in a nude mouse after injection of L540 so it
was advantageous for potential
their apparent cellular characteristics or sensi
work was continued with L540cy only.
No dependency between cultivation time and
cell lines (data not shown).
(A)
Figure 3: Expression of endogenous
(A) Total soluble (40 µg/lane) protein from cell lysates (transferred to a nitrocellulose membraneanti-human PI-9 and GAM-PO (2.1.5fixed, permeabilized (2.4.7) and stained with antidetected by flow cytometry (2.5). mean fluorescence intensities (MFIs) ± SD
The data confirmed the presence of endogenous PI
group and showed that the
expression. These findings are
granzyme B variant (3.5).
3.2 IDENTIFICATION OF MU
At the molecular level a reversible Michaelis complex is formed
inhibitors, followed by a 1:1 covalent complex whereby a covalent bond is
chain oxygen atom of GbS195
mature granzyme B) and the carbonyl carbon of the PI
conformational changes occur in both proteins after covalent binding, it was important to refer to a
The effect of the amino acid exchanges described above on the
sensitivity towards PI-9 was evaluate
both generated prior to this work
malignancies has been demonstrated before
cytotoxic effects on the HL target cell line L540.
cytotoxicity and apoptosis could be shown on L540 as well as L540cy
the protein granzyme B lacking a cell
structures are displayed in Figure
(A)
(B) I
Figure 4: Overview of initial granzyme B
and after Enterokinase cleavage (B)
(A) Schematic structure of the binary eukaryotic expression vectorH22(scFv) and EGb. Igκ: signal peptide sequencecleavage site, Gb: granzyme B encoding sequence, scFv: single chain variable fragment, tag, IVS/IRES: synthetic intron and internal ribosome entry site, Mutated granzyme B sequences could be inserted via expressed in HEK293T cells and purified from supernatant via chromatography (2.4.4). Activation of the protein was (2.4.4) resulting in higher running band in presenceseparated by SDS-PAGE and stained with coomassie(NEB); II: (E)Gb-Ki4(scFv), M: Pre-stained broad range marker in kDa (NEBmarker in kDa (NEB).
amino acid exchanges described above on their functionality as well as their
9 was evaluated using the initial constructs Gb-H22(scFv) and Gb
both generated prior to this work. The cytotoxic potential of Gb-H22(scFv) against CD64
malignancies has been demonstrated before [138]. Gb-Ki4(scFv) was cloned earlier
cytotoxic effects on the HL target cell line L540. In this study, the protocol was optimized
apoptosis could be shown on L540 as well as L540cy cells (Figure
B lacking a cell-binding ligand was prepared (Gb). The corresponding
Figure 4A.
II III
granzyme B constructs (A) and SDS-PAGE of the purified fusion proteins before
(B).
Schematic structure of the binary eukaryotic expression vector pMS encoding for EGbIgκ: signal peptide sequence (L) of immunoglobulin kappa light chain, ECS: enterokinase
B encoding sequence, scFv: single chain variable fragment, ron and internal ribosome entry site, EGFP: enhanced green fluorescent protein.
B sequences could be inserted via XbaI/ BlpI cloning site. (B) expressed in HEK293T cells and purified from supernatant via IMAC and subsequent gel filtration
. Activation of the protein was achieved via incubation with 0.02resulting in higher running band in presence (a) and lower bands in absence (b)
PAGE and stained with coomassie. I: (E)Gb-H22(scFv), M: Protein broad range marker in kDa stained broad range marker in kDa (NEB), III: (E)Gb, M: Protein broad range
(A) Schematic structure of the prokaryotic expression vector pMT encoding for PIsequence for periplasmic localizatioNotI cloning site. (B) Recombinant PIsubsequently via anion exchange chromatography (MonoQ column) and finally via gel filtrationchromatography (GF) (2.4.4). the band of active PIfurther experiments. The band below the 42assays (C,III). (C) To demonstrate the wild-type Gb-H22(scFv) for 1 h at 37°C transferred to a nitrocellulose membrane (western blot using anti-human Gb ((2.1.5); 1: Gb-H22(scFv), 2: Gb-H22(scFv)complex are indicated in grey, full-length protein and complex (red rectangle) thereof in blackbroad range marker in kDa (NEB).
Preparation of recombinant PI-9 and functionality determination via complex formation with
Schematic structure of the prokaryotic expression vector pMT encoding for PI-9. pelBlocalization, H: 6x histidine tag, PI-9: PI-9 encoding sequence, inserted via
Recombinant PI-9 was expressed in E. coli (2.4.3) and purified subsequently via anion exchange chromatography (MonoQ column) and finally via gel filtration
the band of active PI-9 protein is indicated by an arrow. Fraction 6 was used for and below the 42 kDa band was inactive protein, as prove
the functionality of the produced recombinant PI-9 at 37°C (2.7.1.4) before the protein mixture was separated by SDS
transferred to a nitrocellulose membrane (2.4.2). Complexed and uncomplexed protein was detected in a human Gb (I), anti-human PI-9 (II) or anti-His monoclonal antibodies
(scFv) and PI-9, 3: PI-9. Gb cleaved during purification and the corresponding length protein and complex (red rectangle) thereof in black
9 could be produced by bacterial expression and is essential to determine
proteolytic activity of the new granzyme B mutants in presence of PI-9 (3.6.1).
ESTABLISHMENT OF A TEST SYSTEM FOR GRANZYME B MUTANTS
promising mutant, able to induce apoptosis within PI
based on PI-9-positive and –negative cell lines had to be
sis different strategies were pursued: First, the generation of transiently transfected
-9 or the delivery of recombinant granzyme B protein
(A) 33 nM Gb-Ki4(scFv) was incubated with Apoptotic effects were measured via Annexincells; bottom right: early apoptosis; top right: late apoptosis/dead cells. Numbers in the quadrants represthe percentage of cells of each category.treated (+) or untreated (-) cells was separated by SDSmembrane (2.4.2). PI-9-Gb-Ki4(scFv) complex and uncomplexed Gb(2.4.2) using anti-human Gb and GAMin L428 cells. M: Pre-stained broad range marker in kDa (NEB).
In conclusion, the CD30-positive cells
the cytotoxic potential of wild-type and mutant granzyme
lysed after treatment with Gb-Ki4(scFv). Despite the non-specifically detected band at the same
Ki4(scFv) the inactivated SDS-stable granzyme B
rn blot analysis (Figure 7B) in contrast to the findings in PI
(B)
PI-9 on apoptotic activity of Gb-Ki4(scFv).
was incubated with PI-9-negative L540cy and PI-9-positive L428 cellsApoptotic effects were measured via Annexin V/ PI assay (2.7.2.1). Bottom left: viable cells; top left: necrotic cells; bottom right: early apoptosis; top right: late apoptosis/dead cells. Numbers in the quadrants represthe percentage of cells of each category. (B) Total soluble protein from cell lysates (40 µg/lane) of
) cells was separated by SDS-PAGE (2.4.1) and transferred to a nitrocellulose Ki4(scFv) complex and uncomplexed Gb-Ki4(scFv) was detected in a western blot
human Gb and GAM-PO (2.1.5). Unspecific band was detected with equal size stained broad range marker in kDa (NEB).
positive cells used turned out to be suitable for the intended
type and mutant granzyme B on PI-9-positive and
The results were confirmed during ex vivo studies on patient-derived leukemic cells (obtained from
the UK Aachen Dr. Edgar Jost) using CD64-targeting Gb-H22(scFv) and its mutants
positive L428 cells (both HL) for 48 h. Bottom left: viable cells; top left: necrotic
cells; bottom right: early apoptosis; top right: late apoptosis/dead cells. Numbers in the quadrants represent (B) Total soluble protein from cell lysates (40 µg/lane) of Gb-Ki4(scFv)
) and transferred to a nitrocellulose Ki4(scFv) was detected in a western blot
3.6 IN VITRO FUNCTIONAL CHARACTERIZATION OF WILD-TYPE AND MUTANT
GB-H22(SCFV) AND GB-KI4(SCFV)
The main challenge during generation of granzyme B mutants was to avoid a negative effect of the
mutations on the active site of the enzyme and its tertiary structure. To exclude hampering of
functionality, the wild-type and mutated granzyme B-based fusion proteins were tested in
comparative assays regarding their enzymatic activity, their binding affinity, and their specific cell
cytotoxicity.
3.6.1 Proteolytic activity of granzyme B in absence and presence of PI-9
The influence of the single point mutations on the granzyme B proteolytic activity was determined
via a colorimetric substrate assay based on the synthetic granzyme B substrate Ac-IETD-pNA, which
mimics the cleavage and activation site of human procaspase-3 (2.7.1.1). Results for Gb-Ki4(scFv) and
GbR201K-Ki4(scFv) are shown in Figure 8.
0 10 20 30 40 50 600.0
0.1
0.2
0.3
0.4
0.5
0.6
GbR201K-Ki4Gb-Ki4
Time [min]
E40
5 n
m
Figure 8: Enzymatic activity of Gb-Ki4(scFv) and GbR201K-Ki4(scFv).
The proteolytic activity of activated protein was measured via a colorimetric assay based on the synthetic granzyme B substrate Ac-IETD-pNA (2.7.1.1). Reaction was documented for 1 h with a 2 min interval at 37 °C and 405 nm in an Elisa plate reader. Graphs were plotted using GraphPad Prism 4.0.
No differences were observed regarding the measured enzymatic kinetics so it could be concluded
that the active site was not affected by these mutations.
Enzymatic activity of Gb-H22(scFv) and GbR201K-H22(scFv) was additionally quantified via Michaelis-
Menten kinetics. KM values were comparable for both constructs with 104 ± 32 µM for the wild-type
and 105 ± 13 µM for the mutant.
A comparative overview of the activity of all generated mutants, including GbK27A, a mutant
published before and supposed to bind less efficiently to PI-9 (1.4.1), is depicted as grey bars in
Figure 9. Except for the double mutant R28A/R201A and the single mutant R201E, no significant
differences were measured in comparison to the wild-type protein.
Gb-H22
GbK27
A-H22
GbR28
A-H22
GbR28
E-H22
GbR28
K-H22
GbR28
1A/R
201A-H
22
GbR20
1A-H
22
GbR201E
-H22
GbR20
1K-H
22
0.000
0.005
0.010
0.015
0.020
0.025
0.030
0.035without PI-9with PI-9
E40
5nm
/min
Figure 9: Enzymatic activity of Gb-H22(scFv) wild-type and its mutants in the presence and absence of PI-9.
Gb-H22(scFv) was incubated either with our without recombinant PI-9 according to 2.7.1.4. The proteolytic activity was measured via a colorimetric assay based on the synthetic granzyme B substrate Ac-IETD-pNA (2.7.1.1, 2.7.1.4). The graph shows means ± SD of 3 to 6 independent experiments [149].
To confirm the earlier obtained in silico findings (3.23.2) in vitro, the proteolytic activity of wild-type
and mutant granzyme B was analyzed after challenge with recombinant PI-9 in a comparative
enzymatic assay. In the presence of PI-9, GbR28K retained 76 % of its original activity, and GbR201A
retained 46 %. The enzymatic activity of GbR201K was almost the same in the presence or absence of
PI-9 (6 % reduction after PI-9 challenge), so this variant was the least inhibited variant according to
this assay. The catalytic activity of the other mutated granzyme B variants decreased relative to wild-
type granzyme B, or was even lower in the case of the double mutant. Their remaining activity after
complexing ranged from 0.5 % for the double mutant (R28A-R201A) to 25 % for R28E. An exception
was R28A with a remaining activity of 54 % as opposed to the computed predictions [149]. Even
though the result of the latter was promising, this variant was not further tested since it did not show
good results during preliminary cytotoxicity studies on PI-9—positive and –negative cells (data not
shown). The PI-9 insensitivity of the earlier described granzyme B variant K27A (see above) could not
be reproduced either during the in silico or during the in vitro experiments.
The most promising constructs with retained proteolytic activity after incubation with recombinant
PI-9, GbR28K, GbR201A and GbR201K, were further tested, always in comparison to GbK27A and Gb
Representative results for Gb-Ki4(scFv) and GbR201K
(A)
(B)
Figure 10: Binding analysis of granzyme B
50 nM Gb-based constructs were incubated with binding was detected with anti-His Alexa488 (GbR201K-Ki4(scFv) on CD30-positive L428 and CD30GbR201K-H22(scFv) on CD64-positive HL60 and
accumulation of fluorescent protein to bind and internalize allowing it to be visualized via confocal
microscopy. Internalization and binding was also shown for CD64-positive HL60 cells after 2 h
incubation with H22(scFv)-SNAP-BG-647 (Figure 11). Results are depicted in Figure 11.
Construct Cell line 37 °C 4 °C 37 °C (-)
Ki4
(scF
v)-
SN
AP
-BG
-64
7
L540cy
L428
K562
H2
2(s
cFv
)-S
NA
P-
BG
-64
7
HL60
Figure 11: Internalization analysis of Ki4(scFv)-SNAP-BG-647 and H22(scFv)-SNAP-BG-647 into corresponding
target cell lines.
50 nM fluorescent fusion protein or buffer only (-) were incubated with 4*105 target cell lines at 37 °C or 4 °C and internalization was detected with confocal microscopy after 2 h for L540cy, L428, L1236 and HL60 and after 4 h for K562 (2.6). The (scFv)-SNAP-BG-647 signal (upper left), a transmitted light picture (upper right) and the overlay of the two (lower left) are shown.
External binding only was observed at 4 °C; expression level signals differed in intensity depending on
the receptor used. Incubation at 37 °C led to receptor-mediated endocytosis in all cell lines tested
(Figure 11). The more intense fluorescent signal observed for H22(scFv)-SNAP-BG-647 was probably
due to a better coupling efficiency of the SNAP-based protein.
Figure 12: Cytotoxic effects of Gb-Ki4(scFv) and Gb-H22(scFv) wild-type and their mutants respectively on
PI-9-negative target cell lines.
L540cy and stimulated (50 U/mL IFNγ) HL60 cells were incubated for 72 h at 37 °C with decreasing protein concentrations (serial dilution 1:3) of Gb-Ki4(scFv) and its mutants or Gb-H22(scFv) and its mutants, respectively. Cell viability was evaluated with a colorimetric XTT-based assay (2.7.3). Viability of untreated cells was set to 100 %. The graphs show means ± SD of triplicates per dilution. IC50 values were evaluated with GraphPad Prism 4.0 and are listed for all constructs.
Due to the absence of PI-9 in L540cy and HL60 cells, no differences of functionality should occur
during the performed assays. This could be confirmed by the dose-depending curves whose values
were determined after incubation of the target cells with the fusion proteins Gb-Ki4(scFv) and
Gb-H22(scFv) and their mutants. The IC50 values (between 1.7 and 5.1 nM or 1.2 and 5.3 nM,
respectively, summarized in Figure 12) were all in the same range not differing significantly from the
wild-type. No cytotoxic effects were determined on target receptor negative cell lines HL60 for
Gb-Ki4(scFv) constructs and Ramos for Gb-H22(scFv) constructs (data not shown).
The specificity of cell death induced by GbR201K-H22(scFv) was additionally confirmed by a
competitive assay (2.7.3). Therefore cells were incubated with 10 nM of the fusion protein in the
absence and presence of 10 or 100 nM H22(scFv). 48 h incubation of HL60 cells with
GbR201K-H22(scFv) alone resulted in a remaining cell viability of 44 % compared to 100 % of
untreated cells determined via XTT assay. Cytotoxic effects were significantly reduced by 14 % or
25 % in presence of 10 nM H22(scFv) or 100 nM H22(scFv), respectively. Incubation with H22(scFv)
alone did not reduce cell viability significantly (data not shown).
3.6.5 Apoptotic activity on PI-9-negative cell lines
To verify apoptosis induction as underlying mechanism of the observed cell death, an Annexin V/ PI
assay was performed as described in 2.7.2.1. Results are depicted in Figure 13.
(A)
Buffer
Gb-Ki4
GbK27
A-Ki4
GbR28
K-Ki4
GbR20
1A-K
i4
GbR201K
-Ki4
0
25
50
75
100 L540cy
Via
bilit
y [%
]
(B)
Buffer
Gb-H22
GbR201K
-H22
0
25
50
75
100 HL60K562
Via
bilit
y [%
]
Figure 13: Apoptotic effects of Gb-Ki4(scFv) or Gb-H22(scFv) wild-type and their mutants respectively on PI-9-
negative target cell lines.
21 nM CFPs were incubated with target cell lines for 48 h at 37°C. Apoptotic effects were measured via Annexin V/ PI assay (2.7.2.1), normalized to buffer control and converted to viability. The bar graphs show means ± SD of three independent experiments. (A) Result after incubation of Gb-Ki4(scFv) and its mutants with CD30+ L540cy cell line. (B) Result after incubation of Gb-H22(scFv) and its mutants with stimulated (50 U/mL IFNγ) CD64+ HL60 cell line. CD64 K562 cell line was used as negative control.
Figure 14: Cytotoxic effect of Gb-Ki4(scFv) and its
11 nM CFPs were incubated with PICell viability was evaluated with a colorimetric XTT100 %. The bar graphs show means ± SD of three to five determined via two-tailed t-test, (*)
As depicted in Figure 14, GbK27A
GbR201A-Ki4(scFv) showed significantly
GbR201K-Ki4(scFv), reduced the viability to 62
(CML) compared to the untreated control (p
3.7.2 Apoptotic activity on PI
Induction of apoptosis within all three cell lines was
mutant GbR201K-Ki4(scFv) by activating
Here, a statistically significant increase of the relative caspase
effects induced by the wild-type protein was detected.
11 nM CFPs were incubated with PIApoptosis was evaluated by caspaseof Gb-Ki4(scFv). The bar graphs show means ± SD of three to five independent experiments. significance was determined via two
In summary, as predicted from the
promising mutant in all performed
3.8 IN VIVO BIOLOGICAL ACTIVITY O
3.8.1 Stability of Gb-Ki4(scFv) constructs in mouse serum
The stability of Gb-Ki4(scFv) and Gb
prior to conducting in vivo
determination of residual binding activity.
fluorescence-labeled antibodies specific for both the N
anti-His Alexa 488) were used. Exemplary results are shown in
For both constructs, around 90
37 °C whereas the binding activity of the granzyme
Ki4(scFv) and GbR201K-Ki4(scFv) on PI-9 positive target cell lines.
PI-9+ HL cell lines L1236 and L428 or PI-9+ CML cell line K562 for 48Apoptosis was evaluated by caspase 3/7 assay (2.7.2.2). Data are shown as normalized to
The bar graphs show means ± SD of three to five independent experiments. significance was determined via two-tailed t-test, (*): p<0.05, (***): p<0.001 (2.10).
as predicted from the in silico calculations, GbR201K-Ki4(scFv) was found to be the most
promising mutant in all performed in vitro studies, so it was further tested in vivo
IOLOGICAL ACTIVITY OF GB-KI4(SCFV) AND GBR201K
Ki4(scFv) constructs in mouse serum
Ki4(scFv) and GbR201K-Ki4(scFv) in mouse serum compared to PBS
studies. This was accomplished indirectly by
determination of residual binding activity. To verify stability of the full-length fusion protein
labeled antibodies specific for both the N- as well as the C-terminus (anti
Exemplary results are shown in Figure 16.
% binding activity was detected after incubation for 48
°C whereas the binding activity of the granzyme B constructs incubated in serum decreased to
h. However, within the first 10 h of incubation, a reduction of only 10
Figure 16: Serum stability of Gb-Ki4(scFv) and GbR201K
70 ng/µL of each fusion protein wasafter different time points (2.4.8). Ranti-His Alexa 488 (2.5.1). Data exemplary of percentage serum and PBS stability
Figure 17: Evaluation of the established L540cy and L428 tumor models.
(A) L540cy-tumor was resected from sacrificed animals and 8 µm cryo-sections were prepared in a freezing microtome. Endothelial cells were stained pink with alkaline phosphatase without levamisole to determine vascularization of the tumor (2.8). (B) Blood serum was isolated from tumor-bearing mice and CD30 concentration was quantified via an ELISA assay (2.4.9). The results were correlated to the corresponding tumor sizes. M1, M2 and M3: three different mice determined. (C) 0.75 nM Ki4(scFv)-SNAP-BG-747 was injected into an L540cy-tumor bearing mouse. Measurement was performed with deep red filter set (730–950 nm), exposure time 500 ms. I) background signal, II) image with fluorescence signal (yellow, Ki4(scFv)-SNAP-BG-747). Receptor depending targeting of the tumor (left side) was detected after 6 h via in vivo molecular imaging with the CRi-Maestro system (2.9.4). Unspecific accumulation of the 747 signal in the kidney was observed (right side) as well as retained signal at the injection site (right eye). (D) 5*106 Katushka transfected L428 cells resuspended in 50 % Matrigel™ were injected subcutaneously into the right hind limb (2.9.3). Tumor area was detected and calculated via in vivo molecular imaging with CRi-Maestro system (2.9.4). Measurement was performed with yellow filter set (630–850 nm), exposure time 500 ms. I) fluorescence image with red circled tumor area of one representative mouse, II) same picture as I, edited to visualize the mouse.
(A) Treatment schedule indicating intravenous (i.v.) injection of into the right hind limb (2.9.3), protein injection and days of measurement. (B) GbR201K-Ki4(scFv) (N = 6) or with nonwith caliper and related to initial tumor test, (***): p < 0.001 (2.10). (C) L540cand cut into 8 µm sections in a freezing microtome. CD30 receptor Ki4(scFv)-SNAP-BG-Vista-Green (2.8
The data obtained clearly indicate
successfully triggered an arrest of tumor growth, whereas tumors in mice receiving non
GbR201K-H22(scFv) more than doubled in size
3.8.4 Comparative in vivo
positive L428 cells
The established model based on Kat2 transfected L428 cells (
anti-tumor activity against L540cy-induced tumors.
(A) Treatment schedule indicating intravenous (i.v.) injection of 5*106 L540cy cells resuspended in Matrigel, protein injection and days of measurement. (B) Mice were treated either with
6) or with non-binding control GbR201K-H22(scFv) (N = 3). Tumor sizecaliper and related to initial tumor size (100 %). Statistical significance was determined via two
L540cy-tumor was resected from sacrificed GbR201K-Ki4(scFv) a freezing microtome. CD30 receptor expression was visualized by
2.8) and confocal microscopy (2.6).
clearly indicates that treatment with GbR201K-Ki4(scFv) was CD30
triggered an arrest of tumor growth, whereas tumors in mice receiving non
doubled in size (mm²) (p < 0.001).
effects of Gb-Ki4(scFv) and GbR201K-Ki4(scFv)
he established model based on Kat2 transfected L428 cells (3.8.2) and molecular imaging (
o compare the therapeutic efficacy of the most promising mutant GbR201K
against PI-9-positive tumors. 21 mice were divided
3 groups and injected with cells as described above (
GbR201K-Ki4(scFv), administered
Figure 19A. GbR201K-H22(scFv) was used as
Only the mutant GbR201K-Ki4(scFv)
reduction was statistically significant compared to both wild
whereas the difference between wild
statistically significant (Figure 19
(A)
(B)
Figure 19: Evaluation of the in vivo
(A) Treatment schedule indicating intravenous (i.v.) injection of in Matrigel™ into right hind limb (2.9.3either with GbR201K-Ki4(scFv) (N =(N = 7). Tumor size was measured viarelated to initial tumor size (100 %). (**): p < 0.005, ns: not significant (2.10and cut into 8 µm sections on a freezing microtome. Ki4(scFv)-SNAP-BG-Vista-Green (2.8Kat2 signal from transfected cells, III)
injected with cells as described above (3.8.2). Treatment with 50
administered i.v., was started one day after injection of cells
H22(scFv) was used as the non-binding control.
Ki4(scFv) killed the PI-9-positive cells in vivo. Here, the tumor size
reduction was statistically significant compared to both wild-type (p < 0.05) and control (p
whereas the difference between wild-type induced tumor size decrease and control group was not
19B).
(C)
I
anti-tumor activity against L428-induced tumors.
(A) Treatment schedule indicating intravenous (i.v.) injection of 5*106 Kat2-transfected L428 cells2.9.3), protein injection and days of measurement. (B) = 7), Gb-Ki4(scFv) (N = 7) or with non-binding control GbR201K
size was measured via in vivo molecular imaging with the CRi-Maestro System (%). Statistical significance was determined via two-tailed t
2.10). (C) L428-tumor was resected from sacrificed controlµm sections on a freezing microtome. CD30 receptor expression was visualized by staining with
2.8) and confocal microscopy (2.6): I) Ki4(scFv)-SNAP-BGKat2 signal from transfected cells, III) overlay of an enlarged image.
CMML I and CMML III, PI-9 was detected at time point 0 as well, albeit significantly lower than after
14 or 24 h respectively (data not show
positive. Results for all determined cells are summarized in
PI-9-negative did not even express PI
(A)
Figure 20: Expression of endogenous PI
CMML or AMML patients (2.1.8).
(A) Exemplary data for patient CMMLfor different time intervals, before harvesting. separated by SDS-PAGE (2.4.1) and transferred to a nitrocellulose membrane. Endogenousa western blot (2.4.2) using anti-human PI(NEB); Pos.: recombinant PI-9 as positive control. patient cells after at least 14 h cultivation duration.
In summary, three out of the four investigated CMML samples and one out of the three determined
AMML samples exhibited PI-9 expression after 5 days in culture.
3.9.2 Receptor phenotyping of PBMCs
So far it is not clear if the receptor CD64 is
overexpression on the isolated cells and to determine its co
markers for AMML and CMML (CD56, CD33 and CD14, compare
accordingly by flow cytometry and confocal microscopy
shown in Figure 21A.
All patient samples showed a high CD64 expression ranging from 56 to 98
staining revealed that in most patients co
occurred. Co-expression of CD64 and CD14 also became evident during the investigations.
and C confirm the double staining of cells during confocal micro
CMML samples exhibited expression of PI-9 after at least 14 h of incubation in R10 medium. In
9 was detected at time point 0 as well, albeit significantly lower than after
h respectively (data not shown). Only one AMML probe (AMML I) turned out to be PI
ll determined cells are summarized in Figure 20B, whereby the cells indicated as
negative did not even express PI-9 after 5 days in supplemented RPMI medium
(B)
Patient
CMML I CMML II CMML III CMML IV AMML I AMML II AMML III
Expression of endogenous PI-9 (2.4.7) in primary leukemic cells derived from peripheral blood of
) Exemplary data for patient CMML IV. Cells were incubated in R10 in presence or absence of 200before harvesting. Total soluble protein from cell lysates
) and transferred to a nitrocellulose membrane. Endogenoushuman PI-9 and GAM-PO (2.1.5). M: Pre-stained broad range marker in kDa
9 as positive control. (B) Overview of PI-9 expression for all determined primary ivation duration.
In summary, three out of the four investigated CMML samples and one out of the three determined
9 expression after 5 days in culture.
henotyping of PBMCs
if the receptor CD64 is a valuable target for AMML and CMML. To investigate its
expression on the isolated cells and to determine its co-expression with the known tumor
markers for AMML and CMML (CD56, CD33 and CD14, compare 1.6), the cells were phenotyped
by flow cytometry and confocal microscopy. An overview of the flow cytometric r
All patient samples showed a high CD64 expression ranging from 56 to 98 % on all cells. Double
staining revealed that in most patients co-expression of CD64 and CD33 as well as CD64 and CD
expression of CD64 and CD14 also became evident during the investigations.
and C confirm the double staining of cells during confocal microscopy (2.6). Here, cells from patient
I clearly showed double staining via anti-CD56-APC and anti-CD64-FITC (
III double staining with anti-CD56-APC + anti-CD64-FITC, anti-CD33-APC + anti
PBMCs were isolated from peripheral blood of leukemic patients (CMML or AMML, 2.1.8) and incubated with specific antibodies (2.1.5) according to manufacturer’s instructions (2.5.3). Binding was detected by flow cytometry (2.5.3) or confocal microscopy (2.6) (A) Overview of expression profile of patient-derived primary leukemic cells determined via flow cytometry; %: portion of receptor positive cells within the whole isolated cell population; n.d.: not determined; numbers in brackets: strong CD64 expression detected. (B-C) Confocal microscopy of cells from patient CMML I (B) and CMML III (C) stained with antibodies (2.1.5) as indicated.
As a result of the described observations, targeting AMML and CMML patient-derived cells and
potentially killing them with the CD64-specific H22(scFv)-based CFPs was pursued.
3.9.3 Target cell binding and internalization
After detection of CD64 expression, specific binding of the H22(scFv)-derived fusion proteins was
likewise determined. Exemplary results are shown in Figure 22A for cells from patient CMML IV after
incubation with Gb-H22(scFv), GbR201K-H22(scFv) or H22(scFv)-ETA’, respectively. All constructs
Specific CD64-based internalization was demonstrate
H22(scFv)-SNAP-BG-647 or H22(scFv)
CMML III and CMML IV in Figure
indicating that no unspecific binding occurred to CD64
(A)
I
(B) I
Figure 22: Binding and internalization a
(A) 50 nM H22(scFv)-fused constructs were incubated with peripheral blood of patient CMML IV for 30(2.1.5) and flow cytometry (2.5.1). incubated with 4*105 CD64-positive primary cells from patient 10 min at 37 °C and internalization was detected with c
GbR201K-H22(scFv) to evaluate differences in their efficacy depending on
targeted cells. The PI-9 insensitive
viability was measured via XTT
Prior to cytotoxic studies, cells were stimulated with IFNγ to retain activation of the cells, which was
confirmed via flow cytometry using CD64
did not induce apoptosis within the targeted cells (data not shown).
CMML
(A)
(B)
Figure 23: Cytotoxic and apoptotic
primary leukemic cells.
21 nM fusion protein was incubated with stimulated (200peripheral blood (2.1.8) of CMML (leftevaluated by XTT metabolization (A) ((2.7.2.1) (B), normalized to buffer control and converted to viabilityindependent experiments. For CMMLH22(scFv)-ETA’. Statistical significance was determined via two
Primary cells were incubated with the CFPs Gb-H22(scFv) and the PI-9
H22(scFv) to evaluate differences in their efficacy depending on PI-
sensitive IT H22(scFv)-ETA’ was used as positive control
XTT (Figure 23A) and Annexin V/ PI staining (Figure
cells were stimulated with IFNγ to retain activation of the cells, which was
confirmed via flow cytometry using CD64-specific antibodies. Without the addition of IFNγ, the CFPs
did not induce apoptosis within the targeted cells (data not shown).
AMML
and apoptotic effects of Gb-H22(scFv), GbR201K-H22(scFv) and H22(scFv)
nM fusion protein was incubated with stimulated (200 U/mL IFNγ) primary mononuclear cells derived from ) of CMML (left side) or AMML patients (right side) for 48 h at 37°C. Viability was
by XTT metabolization (A) (2.7.3) and apoptotic effects were measured via Annexin, normalized to buffer control and converted to viability. The bar graphs show means ± SD of three
For CMML I, AMML I and AMML II no experiments were performed with Statistical significance was determined via two-tailed t-test, (*): p<0.05, (***)
Figure 24: Cytotoxic effects of Gb-H22(scFv) and GbR201K-H22(scFv) on primary leukemic cells of AMML I.
Stimulated (200 U/mL IFNγ) target cells isolated from peripheral blood (2.1.8) were incubated for 72 h at 37 °C with decreasing protein concentrations (serial dilution 1:3) of Gb-H22(scFv) (grey curve) and GbR201K H22(scFv) (black curve). Cell viability was evaluated with a colorimetric XTT-based assay (2.7.3). The graphs show means ± SD of triplicates per dilution.
The determined sigmoidal viability curve confirmed specific and dose-depending cell death for
AMML I with superior efficacy of the mutant. The IC50 value for GbR201K-H22(scFv) was 8.4 nM.
3.9.5 Specificity of target cell death
Since the viability and apoptosis assays were performed with the entire cell population isolated from
blood after Ficoll gradient, it had to be confirmed that cell death was exclusive for malignant cells.
Different approaches were followed.
The challenge here is that positive selection for CD64 expression (either prior to treatment for pre-
selection of only the target cells or after treatment to identify specific reduction of CD64-positive
cells) was not possible, because limited binding of the CD64-specific antibody was observed on cells
after incubation with non-toxic H22(scFv)-binding constructs (data not shown).
Therefore indirect approaches based on flow cytometric analysis had to be evaluated to confirm
successful elimination of the target cells. The first approach was to identify the location of the target
cell population (R1) within the FCS/ SSC dotplot via a CD64-specific antibody. After treatment with
Gb-H22(scFv) or GbR201K-H22(scFv), this particular population decreased by half which is shown in
Figure 25A for the investigated CMML patients. Representative data for CMML I are shown in Figure
25B where a higher decrease for the mutant than for the wild-type protein was detected.
H22(scFv)-ETA’ detected in FSC/SSC dotplot after flow cytometry
21 nM fusion protein was incubated with stimulated (200peripheral blood (2.1.8) of all determined with CD64-specific antibody the location ofFSC/SSC dotplot (R1) and their reduction evaluated with WinMDISD of three independent experiments for CMML samples. t-test, (**): p < 0.005, (***): p < 0.001 (gated CD64-positive R1 region; portion
The data obtained correspond
Annexin V/ PI measurements (
activity.
Other methods to verify specificity of cell death within the mixed cell population
staining of treated cells with Annexin
significant co-expression with CD64. The latter was chosen accord
however, only gave reliable results for cells from patient CMML
target cancer cell population by Gb-H22(scFv), GbR201K
ETA’ detected in FSC/SSC dotplot after flow cytometry.
nM fusion protein was incubated with stimulated (200 U/mL IFNγ) primary mononuclear cells derived from all determined CMML patients for 48 h at 37 °C. By labeling of mononuclear cells
ic antibody the location of the target cell population was identified via flow cytometry and their reduction evaluated with WinMDI 2.8 (2.5.3). (A) The bar graphs show means ±
SD of three independent experiments for CMML samples. Statistical significance was determined via two0.001 (2.10). (B) Exemplary data shown for treated cells of patient
portion of target cells shown in percentage.
corresponds to the results of the previously obtained
V/ PI measurements (3.9.4). This confirms the specificity of the determined cytotoxic
to verify specificity of cell death within the mixed cell population
Annexin V/ PI and a cell-specific APC-labeled antibody
expression with CD64. The latter was chosen according to the results from
however, only gave reliable results for cells from patient CMML I as shown in
CD56-positive cells were gated so it could clearly be
the amount of apoptotic cells within the CD56-positive target cell population increased after
H22(scFv) and even more significantly after treatment with GbR201K
Figure 26: Reduction of target cancer cell population
specific antibodies (2.1.5).
21 nM fusion protein was incubated with stimulated (200peripheral blood (2.1.8) of CMML patients for 48were labeled in parallel with CD56Dotplots show Annexin V/ PI stained cells after specific CD56cells; bottom right: early apoptosis; top right: late apoptosis/dead cells. Numbers in the quadrants represent the percentage of cells of each category.CD14-specific antibody (anti-CD14-APC,
The third approach to verify specificity
treated cells with certain antibodies
monitoring of the decrease of
phenotyping experiments (Figure
remaining CD14-positive cells after treatment
cells. After incubation with the
Results are displayed as histograms in
to the buffer control was detected
With the help of all described approaches, specificity of cell death within the heterogeneous primary
eduction of target cancer cell population by Gb-H22(scFv) and GbR201K-H22(scFv)
nM fusion protein was incubated with stimulated (200 U/mL IFNγ) primary mononuclear cells derived from ) of CMML patients for 48 h at 37°C. (A) Treated and untreated cells
were labeled in parallel with CD56-specific antibody (anti-CD56-APC, 2.1.5) and AnnexinV/ PI stained cells after specific CD56- gating. Bottom left: viable cells; top left: necrotic
cells; bottom right: early apoptosis; top right: late apoptosis/dead cells. Numbers in the quadrants represent the percentage of cells of each category. (B) Treated and untreated cells of patient CMML II were
APC, 2.1.5). M1 represents cells with strong CD14 expression.
to verify specificity of CD64-selected cell death was accomplished by staining the
certain antibodies which were co-expressed with CD64 (Figure
the decrease of specific receptor expressing cells. For example, for
Figure 21) indicated a co-expression of CD14 and CD64 so
after treatment could be correlated to the targeted
fter incubation with the CFPs, the cells were harvested and labeled with anti
Results are displayed as histograms in Figure 26B. A clear decrease for the treated samples compared
to the buffer control was detected so it could be concluded that cell death was specific.
approaches, specificity of cell death within the heterogeneous primary
cell population induced by the CD64-targeting constructs could be confirmed.
IFNγ) primary mononuclear cells derived from Treated and untreated cells of patient CMML I
) and Annexin V/ PI (2.7.2.1). Bottom left: viable cells; top left: necrotic
cells; bottom right: early apoptosis; top right: late apoptosis/dead cells. Numbers in the quadrants represent Treated and untreated cells of patient CMML II were labeled with
M1 represents cells with strong CD14 expression.
was accomplished by staining the
Figure 21). This allowed
example, for CMML II,
CD64 so the amount of
the targeted CD64-expressing
the cells were harvested and labeled with anti-CD14-APC.
B. A clear decrease for the treated samples compared
so it could be concluded that cell death was specific.
approaches, specificity of cell death within the heterogeneous primary
(A) Schematic structure of the binary eukaryotic expression vector pMS EGm-Ki4(scFv). Ig-κ: signal peptide sequencecleavage site, H: 6x histidine tag, IVS/IRES: synthetic intron and internal ribosome entry site, EGFP: enhanced green fluorescent protein. Enterokinase cleavage site indicated with in HEK293T cells and purified from supernatant via 0.02 U/µg Enterokinase (2.4.4). Protein w(2.4.1) or transferred to a nitrocellulose membrane (2.4.2). Specific protein bands were detected at 61.5kDa (NEB).
As in the case of the granzyme
N-terminus to facilitate expression in eukaryotic cells.
supernatant should allow correct post
glycosylation sites. The protein was purified by affinity chromatography selecting for the His
with a yield of 1 mg/L, as determined by SDS
The identity of the protein was confirmed by western blot analysis using an anti
(2.4.2). Under denaturing conditions, SDS
150 kDa, a band with the expected molecular weight of 61.5
Overview of granzyme M-based constructs (A) and SDS-PAGE and western blot analysis
of the binary eukaryotic expression vector pMS encoding forκ: signal peptide sequence (L) of immunoglobulin kappa light chain, ECS: enterokinase
cleavage site, H: 6x histidine tag, IVS/IRES: synthetic intron and internal ribosome entry site, EGFP: enhanced Enterokinase cleavage site indicated with arrow. (B) Fusion proteins were expressed
in HEK293T cells and purified from supernatant via IMAC (2.4.4). Activation of the protein was done with Protein was separated by SDS-PAGE and either stained with coomassie (I)
) or transferred to a nitrocellulose membrane for western blotting with anti-human Specific protein bands were detected at 61.5 kDa and ~150 kDa. M: Pre-stained broad range marker in
granzyme B-based CFPs, an enterokinase site precedes the granzyme
terminus to facilitate expression in eukaryotic cells. The secretion of the fusion proteins into the
allow correct post-translational modification because granzyme
ation sites. The protein was purified by affinity chromatography selecting for the His
mg/L, as determined by SDS-PAGE (2.4.5) and activated with enterokinase (
The identity of the protein was confirmed by western blot analysis using an anti-
). Under denaturing conditions, SDS-PAGE revealed, apart from a band at approximately
a band with the expected molecular weight of 61.5 kDa (Figure 27B).
Hence, the intended granzyme M constructs could successfully be generated and
lower band (Figure 28A). The same results were obtained for Gm
The enzymatic functionality of the fusion protein was also verified by the applied colorimetric assay.
No activity was detected for the uncleaved variant EGm
dependent increase in extinction was observed for
(A)
Figure 28: Enzymatic activity of Gm
(A) Recombinant PI-9 (3.4) and Gm-was separated by SDS-PAGE and transferred to a nitrocellulose membrane (was detected in a western blot using antiin kDa (NEB). (B) The proteolytic activity of activated protein was measured via a colorimetric assay based on the synthetic granzyme M substrate Suc405 nm for different concentrations as indicated and compared to uncleaved variant EGmgraphs show means ± SD of three independent experiments.
These results confirm that enzymatically
expression in HEK293T cells and subsequent enterokinase digestion
Functional characterization of granzyme M fusion proteins
after enterokinase cleavage was tested by two methods, one based on the
to cleave PI-9 (2.7.1.3), and the other one using a colorimetric assay with a
, Suc-Ala-Ala-Pro-Leu-pNA (2.7.1.1).
human PI-9 confirmed the cleavage of PI-9 after 24
H22(scFv) in a 3:1 molar ratio. Recombinant PI-9 was partially degraded during purification for
eavage by granzyme M was nevertheless clearly visible, intensifying the
ame results were obtained for Gm-Ki4(scFv) (data not shown).
The enzymatic functionality of the fusion protein was also verified by the applied colorimetric assay.
tected for the uncleaved variant EGm-H22(scFv), whereas a concentration
dependent increase in extinction was observed for active Gm-H22(scFv) (Figure 28
(B)
Buffer
EGm-H
22 1µ
M
Gm-H22
200
nM
Gm-H22
1 µM
Gm-H
22 2
µM0.00
0.25
0.50
0.75
1.00
Abs
orba
nce
405n
m
Gm-H22(scFv) tested by cleavage of PI-9 or a colorimetric substrate
-H22(scFv) were incubated for 24 h at 37 °C (2.7.1.3) before protein mixture PAGE and transferred to a nitrocellulose membrane (2.4.2). Active and inactivated PI
was detected in a western blot using anti-human PI-9 and GAM-PO (2.1.5) M: Pre-stained broad range marker oteolytic activity of activated protein was measured via a colorimetric assay based on
substrate Suc-Ala-Ala-Pro-Leu-pNA (2.7.1.1). Reaction was documentednm for different concentrations as indicated and compared to uncleaved variant EGm
graphs show means ± SD of three independent experiments.
enzymatically active granzyme M fusion proteins could be produced via
K293T cells and subsequent enterokinase digestion.
specific binding of all granzyme M-based CFPs was tested by flow cytometry using the
target cell lines. Exemplary results are shown for Gm-H22(scFv) targeting human
positive AML cell line HL60 (Figure 29A) and CD64-positive primary cells from a CMML patient
negative L428 cells were used as the control cell line (Figure 29
(A) (B)
H22(scFv).
H22(scFv) was incubated with 4*105 HL60 cells (A) or primary leukemic cells derived from peripheral for 30 min on ice. Binding was detected with anti-His Alexa488 (
L428 cell line was used as negative control.
fluorescence shift was detected on CD64-positive cells, so specific binding could be
based CFP. No binding was observed on the control cell line L428
confirming that the binding activity was selective for the targeted receptor.
(A) Target cells were incubated for 72 Gm-H22(scFv) on stimulated (50 U/mbased assay (2.7.3). Viability of untreated cells was set to 100dilution. CD64- cell line L428 and nonHL60 cells were incubated for 72 h free H22(scFv). Viability was evaluated viathree independent experiments. Statistical significance was determined via two(2.10); ns: not significant. (C) 66 nM G48 h at 37°C. CD64 K562 cell line was used as PI assay (2.7.2.1). Bottom left: viable cells; top left: necrotic cells; bottom right: early apoptosis; top right: late apoptosis/dead cells. Numbers in the quadrants represent the percentage of cells of each category.
Target cells were incubated for 72 h at 37°C with decreasing protein concentrations (U/mL IFNγ) HL60 cells. Cell viability was evaluated with a colorimetric XTT
Viability of untreated cells was set to 100 %. The graphs show means ± SD of triplicates per non-stimulated HL60 cells were used as control. (B) Stimulated (50
at 37°C with 10 nM Gm-H22(scFv) in competition with different dilutions of Fv). Viability was evaluated via XTT metabolization (2.7.3). The bar graphs show means
Statistical significance was determined via two-tailed tnM Gm-H22(scFv) was incubated with stimulated (50 U/m
K562 cell line was used as negative control. Apoptotic effects were measured via AnnexinBottom left: viable cells; top left: necrotic cells; bottom right: early apoptosis; top right: late
cells. Numbers in the quadrants represent the percentage of cells of each category.
granzyme B Gm-Ki4(scFv) was tested in comparison to Gb
L428, L1236 and L540cy. The former
resistant against apoptosis induction by wild
PI assay are shown in Figure 31.
Figure 31: Apoptotic effect of Gm-Ki4(scFv) compared to
11 nM fusion protein was incubated with HL cell lines L428 (PIat 37 °C. Apoptotic effects were measured via Annexinnecrotic cells; bottom right: early apoptosis; top right: late apoptosis/dead cells. Numbers in the quadrants represent the percentage of cells of each category
Gm-Ki4(scFv) is able to kill PI-9
L540cy. As expected, Gb-Ki4(scFv) only kills
effectively than Gm-Ki4(scFv). During a dose
for Gm-Ki4(scFv) on L540cy was 272
(Figure 12A). No unspecific toxic effects were determined on CD3
To evaluate the potential of granzyme M with PI-9-positive cells, in comparison to wild
Ki4(scFv) was tested in comparison to Gb-Ki4(scFv) on the CD30
he former of these two are PI-9-positive and have been shown to be
apoptosis induction by wild-type Gb-Ki4(scFv) (Figure 7). Results for an Annexin
Ki4(scFv) compared to Gb-Ki4(scFv).
nM fusion protein was incubated with HL cell lines L428 (PI-9+), L1236 (PI-9+) and L540cy (PI°C. Apoptotic effects were measured via Annexin V/ PI assay (2.7.2.1). Bottom left
necrotic cells; bottom right: early apoptosis; top right: late apoptosis/dead cells. Numbers in the quadrants represent the percentage of cells of each category.
9-positive cells L428 and L1236 and also the PI
Ki4(scFv) only kills the PI-9-negative cell line L540cy but
Ki4(scFv). During a dose-depending cytotoxicity assay the measured IC
Ki4(scFv) on L540cy was 272 nM which is 100x lower than was determined for Gb
A). No unspecific toxic effects were determined on CD30-negative cell lines (data not
To confirm the potential of granzyme M-based CFPs for the treatment of further tumor entities
425(scFv), which target the epidermal growth factor receptor (EGFR)
(A,B) 21 nM fusion protein was incubated with stimulated (200from peripheral blood (2.1.8) of AMML or CMML patients (as indicated) for 48been tested according to 3.9.1. Viability was evaluated by XTT metabolization (A) (were measured via Annexin V/ PI assay (Data for Gm-H22(scFv) were compared to Gbshow means ± SD of three independent experiments.test, (*): p < 0.05 (2.10); n.d.: not determined.peripheral blood (2.1.8) of patient AMML II (PIwith decreasing protein concentrations (serial colorimetric XTT-based assay (2.7.3).
Cytotoxic and apoptotic effects of Gm-H22(scFv) on primary leukemic cells.
nM fusion protein was incubated with stimulated (200 U/mL IFNγ) primary mononuclear cells derived ) of AMML or CMML patients (as indicated) for 48 h at 37°C. PI
. Viability was evaluated by XTT metabolization (A) (2.7.3PI assay (2.7.2.1), normalized to buffer control and converted to viability (B).
H22(scFv) were compared to Gb-H22(scFv) and GbR201K-H22(scFv) from Figure show means ± SD of three independent experiments. Statistical significance was determined via two
); n.d.: not determined. (C,D) Stimulated (200 U/mL IFNγ) target cells ) of patient AMML II (PI-9-) (A) and AMML III (PI-9-) (B) were incubated for 72
with decreasing protein concentrations (serial dilution 1:3) of Gm-H22(scFv). Cell viability was evaluated with a ). The graphs show means ± SD of triplicates per dilution.
These data clearly indicated that Gm-H22(scFv) could specifically kill leukemic cells
H22(scFv), it performed as well as the new mutant GbR201K
Abbreviations: GrB, Gb, GzmB: granzyme B; hLHR: human luteinizing hormone receptor; Her2: human epidermal growth factor receptor 2; Kex2: killer expression defective 2; EGFR: epidermal growth factor receptor; VEGF: vascular endothelial growth factor; dsFv: disulfide stabilized Fv fragment; n.d.: not determined in other publications; EK: Enterokinase; PEAII: second domain of Pseudomonas Exotoxin A (residues 253-364); (in vivo): cell line was successful killed in mice; * compare 3.1; # Fpe: furin site sequence from PEA (amino acids 273-282), Fdt: furin site sequence from diphtheria toxin (amino acids 187-196), R9: poly-arginine tract; **as determined in the same study.
4.2 IMPACT OF ENDOGENOUS PI-9 EXPRESSION IN CANCER CELLS ON THE PRO-
APOPTOTIC ACTIVITY OF GRANZYME B
A direct correlation between PI-9 expression and resistance to apoptosis induction by wild-type
granzyme B was verified in this study (Figure 7). Several groups have already determined the
expression of the granzyme B inhibitor PI-9 in primary cancer cells. An overview of the results is
shown in Table 10 and Table 11. The impact of resistance on granzyme B-based immunotherapeutics
is controversial, since some reports claim that resistance to perforin-dependent pathways is not a
relevant escape mechanism in lymphoma [148] and that the induction of apoptosis by granzyme B
can be even achieved in cells expressing or upregulating PI-9 (compare Table 9). For example, Cao et
native cell lysates of PI-9-positive cells with a granzyme M based CFP or by cell lysis and subsequent
western blot analysis of those cells after treatment with a corresponding construct. In fact, the high
potential of this approach is supported by the finding, that the interaction between PI-9 and
granzyme M has a high stoichiometry of inhibition (SI) value of 63, which means that PI-9 is a much
better substrate than inhibitor of granzyme B [56]. By cleaving PI-9, granzyme M could therefore
clear the way for apoptosis induced by granzyme B. Hence, granzyme M could be useful, not only in a
therapeutic context, especially against treatment-resistant cells on its own, but also in combination
with granzyme B.
In contrast to granzyme B, granzyme M has not been investigated as extensively, so in the future
both promising, as well as adverse effects may be detected on its prospective role as
immunotherapeutic. Known so far is e.g. that on the down side, as described for granzyme B, a
specific endogenous inhibitor, called serpin B4, has recently been suggested to form a typical SDS-
stable serpin–protease complex with granzyme M in vitro and thereby inhibited NK cell-mediated cell
death in Hela cells [223]. However, the affinity between the two proteins is of a second-order rate
constant of 1.3 x 104 M-1 s-1 which is two orders of magnitude below that of granzyme B and PI-9 and,
in general, below the range of biologically-relevant interactions. This rather weak inhibition might be
overcome by CFP-related excessive delivery of the effector molecule to the cells. Furthermore, the in
vivo expression of this inhibitor has not been reported and any potential escape mechanisms in
tumors remain to be investigated. Other serpins present in plasma, like α1-antichymotrypsin and α1-
proteinase inhibitor, could also become a limiting factor for granzyme M activity in blood as
suggested earlier [56]. Their inhibitory relevance has to be evaluated in the future in serum-stability
assays, and respective modifications, as shown in this study for granzyme B, should be considered.
Other modifications, also in accordance to granzyme B, might involve the cationic sites within
granzyme M that may lead to off-target effects [221]. Hence, current literature on this topic should
be monitored carefully, as in vivo studies should be pursued to further evaluate the potential of
granzyme M in immunotherapeutic approaches.
Concluding, with the novel granzyme B mutant, identified during in silico homology modeling, it was
demonstrated on in vitro, in vivo as well as ex vivo level that residual therapy-resistant cancer cells
can be killed, provided the resistance is due to PI-9 expression as an escape mechanism. Additionally,
the cytotoxic potential and the PI-9 insensitivity of granzyme M-based fusion proteins was verified in
vitro and ex vivo clearing the way for a potential combination treatment with both granzymes.
Outlook ________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________
111
5 Outlook
The presented data emphasize the great efficacy of granzyme B-based cytolytic fusion proteins (CFPs)
in vitro, in vivo as well as ex vivo and especially verify the necessity and potential of directed
modifications, here to pave the way for inhibitor-insensitive treatment. On this basis further
investigations and optimizations are now ongoing in our group concerning the structure of the
corresponding CFPs as well as their extended application.
Structural modifications include the introduction of an additional modification to reduce the high
positive surface charge of granzyme B (pI ~ 10) which is mainly caused by two major heparan-binding
motifs and might result in off-target effects by intrinsic binding to heparin sulfate proteoglycans. In a
previous study, the basic amino acid residues involved in heparan sulfate binding were substituted
for alanine which did not interfere with ligand binding or enzyme activity and reduced the negative
impact of extracellular matrix effects [224]. Hence, specific cytotoxicity of granzyme B could be
increased by using this granzyme B variant. Furthermore, the improvement of endosomal release
after receptor mediated endocytosis within the target cells by certain adapter sequences is now
being investigated in-house (confidential) which suggest that the engineering of granzyme B-based
CFPs by the insertion of functional adapter elements will improve their therapeutic potential. As an
alternative, a pH-sensitive fusogenic peptide has been introduced recently which increased specific
cytotoxicity compared with the construct lacking the peptide, and demonstrated enhanced cellular
uptake and improved delivery of granzyme B to the cytosol of target cells [166]. In addition, another
promising mutational site is presently evaluated in our group which was also suggested from in silico
determinations to be promising to render granzyme B resistant to PI-9 inhibition. The potential of
structure related modifications of the human molecules, however, introduce novel sequences with
potential antigenicity in vivo which have to be evaluated carefully.
Further structural related improvements which are required for translation into clinical application to
reduce potential immunogenicity include either removal of the His6-tag after IMAC purification or
applying alternative purification strategies like ion exchange chromatography independent of any
tags. In addition, the applied murine Ki4(scFv) should either be humanized or replaced by the human
CD30L, which has been shown promising results in combination with human RNase angiogenin
before [187] to prevent HAMA response.
Regarding the extended application of the CFPs, the economic and up-scaled production of active
granzyme B and granzyme M has to be considered. Therefore the yeast Hansenula polymorpha is
now under investigation as a potential alternative expression system. It harbors the additional
advantage to become independent of expensive enterokinase processing of the granzymes by
insertion of the mating factor α prepro-leader sequence allowing straightforward purification of
Outlook ________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________
112
active granzyme from the culture supernatant based on local Kex2-protease activity in the trans-
Golgi compartment. Furthermore, expression of E. coli is presently explored to increase protein yield
with lower expenses.
The described ex vivo findings need to be verified on additional patient material and evaluated in
terms of personalized medicine. Those investigations will be most valuable in combination with
information on the previous treatment and anamnesis. To further evaluate CD64 as valuable
potential therapeutic target also for CMML and AMML in the clinical it will now be investigated if
CD64 is indeed over-expressed on bone marrow samples of AMML and CMML patients. A
corresponding set-up for immunohistochemical determinations with H22 antibody fragments is
already established.
In addition, further in vivo experiments are planned with the described constructs which include the
establishment of a disseminated tumor model using the AML cell line HL60. The same might be
indicated for Hodgkin lymphoma since for this disease a disseminated model is less artificial than the
subcutaneous tumor model applied in this study. Furthermore, the novel variant GbR201K is now
used in combination with several other binders to kill target cells despite the presence of
endogenous PI-9.
The CFP-based treatment of tumor cells, even in presence of PI-9, was shown to be feasible using the
new effector molecule granzyme M. Further combination experiments are planned to test the
potential of granzyme M to clear the way for granzyme B activity by inactivation of PI-9 within the
target cells. These studies will be performed as soon as an adequate PI-9-positive cell line with both
receptors co-expressed becomes available. In addition, the potential of granzyme M-based CFPs has
to be shown in vivo as well using the xenograft tumor models already established for the
granzyme B-based constructs. For both constructs the species-dependent substrate-specificity has to
be taken into account when these enzymes undergo further studies using other than xenograft
tumor models or the in our groups well established mouse inflammation model [213], especially
when proceeding towards clinical application. These specificities can significantly obscure e.g.
immunogenic or unspecific effects, so special care has to be taken when planning pre-clinical studies.
In accordance to granzyme B, novel variants for granzyme M independent of the plasma serpins α1-
antichymotrypsin, α1-proteinase inhibitor and the endogenous inhibitor serpin B4, if detected in vivo
as well, can also be established. Additionally, a granzyme M variant, which does not bind to α-2-
macroglobulin anymore, is planned to prevent its entrapment in human serum.
The here developed novel constructs may eventually be used in the clinic in combination with
conventional treatment approaches helping to reduce the necessary dose of non-specific drugs,
avoiding adverse effects and promoting long-term remission.
currently established. In addition, a novel tumor model with the PI-9-expressing HL cell line L428 was
established here based on optical in vivo imaging. In these proof-of-principle models, it could be
verified in vivo that only the mutant is able to kill cells both in the presence and absence of the
inhibitor, this in contrast to the wild-type which only killed PI-9-negative L540cy tumor cells in vivo.
This is analogous to ex vivo studies with primary cells from patients with the rare diseases CMML and
AMML. These cells were targeted via the CD64 receptor. The data additionally demonstrated that the
new constructs perform even better than H22(scFv)-ETA’. The differences in apoptotic efficacy
between the applied constructs suggest ex vivo studies to be a predictive screening method for
personalized medicine to enable a patient-specific treatment selection.
In addition, a novel human granzyme-based CFP with a pro-apoptotic moiety based on granzyme M is
firstly described here. This serine protease is known to be able to specifically cleave and inactivate
PI-9. It may be a valuable addition to the palette of human effector molecules, in particular for the
development of personalized medicine in which the optimal cytolytic protein and targeting molecule
combination can be selected on a per-patient basis. Different fusion constructs, comprising
granzyme M targeting CD64- and CD30-positive cancer cell lines were generated, expressed and
purified analogous to the granzyme B-based constructs. During in vitro and ex vivo studies
granzyme M was proved to be a potent human anti-tumor agent (IC50 between 1.2 and 6.4 nM for
Gm-H22(scFv)), even in presence of PI-9. CFPs combining granzyme B and granzyme M offer potential
for improved therapeutic approaches by simultaneously triggering different apoptotic pathways to
circumvent possible overexpression of inhibitors within the target cell or by potentiating granzyme B
activity by inactivating PI-9.
In summary, during this thesis two new effector molecules, granzyme M and a PI-9 resistant variant
of granzyme B, granzyme B R201K, have successfully been generated, produced and evaluated in
vitro, ex vivo and in parts also in vivo in regard to their cytotoxic potential and for use in
immunotherapy to treat leukemic diseases or HL. These effector molecules, either individually or in
combination, also bear clinical relevance for the treatment of solid tumors or other hematological
disorders that potentially express PI-9 or selected PI-9-positive cells under immune surveillance or
tumor therapy.
Literature ________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________
115
7 Literature
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immunotoxin 425(scFv)-ETA' demonstrates anti-tumor activity against disseminated human pancreatic cancer in
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translational modification and specific sites of interaction with substrates and inhibitors. Mol Biol Rep, 2011. 38(5): p. 2953-60.
222. BARTH, S., HUHN, M., MATTHEY, B., SCHNELL, R., TAWADROS, S., SCHINKOTHE, T., et al., Recombinant anti-CD25
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Index of Figures and Tables ________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________
IV
9 Index of Figures and Tables
TABLES
Table 1: List of oligo nucleotides used for cloning and DNA sequencing. ............................................. 21
Table 2: Origin and specificity of antibody fragments. ......................................................................... 23
Table 4: Buffers and media.................................................................................................................... 25
Table 5: Reagents for physical or chemical transfection of mammalian cell lines. .............................. 31
Table 6: Overview of the PI-9 expression profile of different cancer cell lines. ................................... 43
Table 7: Affinity constants and relative receptor expression level of CD30-positive cell lines............. 55
Table 8: Serum stability of Gb-H22(scFv) and GbR201K-H22(scFv) in human patient serum (CMML III)
or PBS. ................................................................................................................................................... 77
Table 9: Overview of different granzyme B-based cytolytic fusion proteins (CFPs) published so far. . 88
Table 10: Overview of PI-9 expression in different carcinomas / melanomas. .................................... 91
Table 11: Overview of PI-9 expression in different lymphomas. .......................................................... 93
FIGURES
Figure 1: X-ray crystal structure of the non-covalent Michaelis complex of A353K Manduca sexta
Figure 6: Preparation of recombinant PI-9 and functionality determination via complex formation
with wild-type Gb-H22(scFv). ................................................................................................................ 49
Figure 7: Influence of endogenous PI-9 on apoptotic activity of Gb-Ki4(scFv). .................................... 51
Figure 8: Enzymatic activity of Gb-Ki4(scFv) and GbR201K-Ki4(scFv). .................................................. 52
Figure 9: Enzymatic activity of Gb-H322(scFv) wild-type and its mutants in the presence and absence
of PI-9. ................................................................................................................................................... 53
Figure 10: Binding analysis of granzyme B-based CFPs. ........................................................................ 54
Figure 11: Internalization analysis of Ki4(scFv)-SNAP-BG-647 and H22(scFv)-SNAP-BG-647 into
Index of Figures and Tables ________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________
V
Figure 12: Cytotoxic effects of Gb-Ki4(scFv) and Gb-H22(scFv) wild-type and their mutants
respectively on PI-9-negative target cell lines. ..................................................................................... 57
Figure 13: Apoptotic effects of Gb-Ki4(scFv) or Gb-H22(scFv) wild-type and their mutants respectively
on PI-9-negative target cell lines. .......................................................................................................... 58
Figure 14: Cytotoxic effect of Gb-Ki4(scFv) and its mutants on PI-9-positive target cell lines. ............ 60
Figure 15: Apoptotic effect of Gb-Ki4(scFv) and GbR201K-Ki4(scFv) on PI-9 positive target cell lines. 61
Figure 16: Serum stability of Gb-Ki4(scFv) and GbR201K-Ki4(scFv). ..................................................... 62
Figure 17: Evaluation of the established L540cy and L428 tumor models. .......................................... 64
Figure 18: Evaluation of the in vivo anti-tumor activity against L540cy-induced tumors. .................... 66
Figure 19: Evaluation of the in vivo anti-tumor activity against L428-induced tumors. ....................... 67
Figure 20: Expression of endogenous PI-9 (2.4.7) in primary leukemic cells derived from peripheral
blood of CMML or AMML patients (2.1.8). ........................................................................................... 69
Figure 21: Phenotyping of primary leukemic cells. ............................................................................... 70
Figure 22: Binding and internalization analysis with primary leukemic cells (2.1.8). ........................... 71
Figure 23: Cytotoxic and apoptotic effects of Gb-H22(scFv), GbR201K-H22(scFv) and H22(scFv)-ETA’
on primary leukemic cells. ..................................................................................................................... 72
Figure 24: Cytotoxic effects of Gb-H22(scFv) and GbR201K-H22(scFv) on primary leukemic cells of
AMML I. ................................................................................................................................................. 74
Figure 25: Specific reduction of target cancer cell population by Gb-H22(scFv), GbR201K-H22(scFv)
and H22(scFv)-ETA’ detected in FSC/SSC dotplot after flow cytometry. .............................................. 75
Figure 26: Reduction of target cancer cell population by Gb-H22(scFv) and GbR201K-H22(scFv)
detected with specific antibodies (2.1.5). ............................................................................................. 76
Figure 27: Overview of granzyme M-based constructs (A) and SDS-PAGE and western blot analysis of