and Multispecific Antibodies

218 Pages·2016·6.2 MB·English

by  Christian Panke

TECHNISCHE UNIVERSITÄT MÜNCHEN

Fakultät für Chemie
Lehrstuhl für Biotechnologie

Bi- and Multi-specific Antibodies

Christian Panke

Vollständiger Abdruck der von der Fakultät für Chemie
der Technischen Universität München zur Erlangung des akademischen Grades eines
Doktors der Naturwissenschaften (Dr. rer. nat.)
genehmigten Dissertation.

Vorsitzende: Univ.-Prof. Dr. K. Lang
Prüfer der Dissertation: 1. Univ.-Prof. Dr. J. Buchner
2. Univ.-Prof. Dr. M. J. Feige

Die Dissertation wurde am 18.08.2015 bei der Technischen Universität München
eingereicht und durch die Fakultät für Chemie
am 28.09.2015 angenommen.

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List of publications

Panke, C., Weininger, D., Haas, A., Schelter, F., Schlothauer, T., Bader, S. et al. (2013).
Quantification of cell surface proteins with bispecific antibodies. Protein Eng Des Sel, 26, 645-
654.

Metz, S., Panke, C., Haas, A. K., Schanzer, J., Lau, W., Croasdale, R. et al. (2012). Bispecific
antibody derivatives with restricted binding functionalities that are activated by proteolytic
processing. Protein Eng Des Sel, 25, 571-580.

R. Castoldi, C. Panke, U. Jucknischke1, N. Neubert, J. Schanzer, R. Croasdale, et al. (2015).
TetraMabs: Simultaneous targeting of four oncogenic receptor tyrosine kinases for tumor growth
inhibition. In preparation.

Naumer M, Sonntag F, Schmidt K, Nieto K, Panke C. et al (2012). Properties of the adeno-
associated virus assembly-activating protein. J Virol. 86 (23), 13038-48.I

I

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Abbreviations

A
Ab antibody
ABC antibody binding capacity
ADC antibody drug conjugates
ADCC antibody-dependent cell mediated-cytotoxicity

B
BiTEs bispecific T-cell engagers or short
BSA bovine serum albumin
BsAbs bispecific antibodies
B-CLL B-cell chronic lymphocytic leukemia

C
C domain constant domain
CDC complement-dependent cytotoxicity
cDNA complementary DNA
CDR complement determining region
CI cell index
CLL chronic lymphocytic leukemia
CPRG chlorophenol-red-(cid:533)-D-galacto-pyranoside
CS coding sequence
CS&T cytometer setup & tracking
Ct value cross-threshold value
CTG Cell Titer Glo
C1q complement 1q

D
dAb single domain antibodies
DAF dual-acting-fragment antigen binding
dbs diabodies
Dig digoxigenin
DNA deoxyribonucleic acid
dsFv disulfide stabilized variable region fragment
dsscFv disulfide stabilized single chain variable region fragment
DVD dual variable domain antibody

E
ECM extracellular matrix
EGFR/HER1/ErbB-1 epidermal growth factor receptor
EMT epithelial–mesenchymal transition
EpCAM epithelial cell adhesion molecules

F
Fab fragment antigen binding
Fc fragment crystallizable
FcRn neonatal Fc receptor
iii
FCS fetal calf serum
FITC fluorescein isothiocyanate
FSC forward-side scatter
Fv variable region fragment
F/P fluorophore to protein ratio

G
G4S glycine serine motif
GOI gene of interest

H
HA hemaglutinin
HC heavy chain
HCC hepatocellular carcinoma
HEMA hydroxyethylmethacrylate
HER2 human epidermal growth factor receptor 2
HER3 human epidermal growth factor receptor 3
HGF hepatocyte growth factor
HKG housekeeping genes
HPLC High-Performance Liquid Chromatography
HyD hybrid detectors

I
ICK intestinal cell kinase
Ig immunoglobulins
IGF-1R insulin-like growth factor 1 receptor

K
K association constant
a
K dissociation constant
d
KEX kinetic extrapolation
kih knobs-into-holes

L
LB agar Luria Bertani agar
LC light chain
LEL/EC2 large extracellular loop

M
mAb monoclonal antibody
MESF molecules of equivalent soluble fluorophore
MFI mean fluorescence intensity
MHC major histocompatibility complex
M molar
MMP matrix metalloproteases
N
NEA non essential amino acids
NHS N-hydroxysuccinimide
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NHS-ES N-hydroxysuccinimide esters
NIST National Institute of Standards and Technology
NK cells natural killer cells
NR conditions non-reducing conditions
NTC non-template control
nd not determined

P
PCR polymerase chain reaction
PE phycoerythrin
PEG polyethylene glycol
PEGylation polyethylene glycolylation
PI3K phosphatidylinositol-4,5-bisphosphate 3-kinase
PI4K phosphatidylinositol-4 kinase
PK pharmacokinetics
PKC protein kinase C
PLK-1 polo-like kinases 1
PMT photomultiplier tube
PS penicillin,streptomycin

Q
QFCM quantitative flow cytometry

R
R conditions reduced conditions
Rac1 ras-related C3 botulinum toxin substrate 1
RNAi RNA interference
RT-PCR reverse transcription polymerase chain reaction
RTCA Real-Time Label-Free Cellular Analysis
RTK receptor tyrosine kinase

S
SCB simple cellular beads
scDbs single chain diabodies
SCNAs somatic copy-number alterations
scFab single chain fragment antigen binding
scFv single chain variable region fragment
SEL/EC1 small extracellular loop
SPR surface plasmon resonance
STD standard deviation

T
taFvs/scFv2 tandem single chain variable region fragment
TEMs tetraspanin enriched microdomains
TM transmembrane
Tspans tetraspanins
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U
uPA urokinase-type plasminogen activation
UWA unified window of analysis

V
V domain variable domain
VCAM-1 vascular cell adhesion molecule 1
VEGF vascular endothelial growth factor
VH domain variable heavy domain
VL domain variable light domain
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Contents

1 Summary ................................................................................................................................................... 1
1.1 FACS based quantitation of cell surface receptors and Tetraspanin screening ................................... 1

2 Introduction .............................................................................................................................................. 3
2.1 From monoclonal to multi-specific antibody formats ......................................................................... 3
2.2 Basic structure and functions of Immunoglobulins ............................................................................. 4
2.3 Aspects of antibody engineering and modification ............................................................................. 7
2.3.1 Immunogenicity ............................................................................................................................ 7
2.3.2 Antigen binding affinity ............................................................................................................... 8
2.3.3 Effector functions ....................................................................................................................... 10
2.3.4 Pharmacokinetics ........................................................................................................................ 11
2.3.5 Internalization ............................................................................................................................. 12
2.3.6 Molecular architecture ................................................................................................................ 14
2.4 The prototype of recombinant antibody molecules: Single chain variable fragment ........................ 15
2.5 Bi-and multispecific antibody formats .............................................................................................. 16
2.5.1 DAF-CrossMab: Old players in new format .............................................................................. 20
2.5.2 IgG-dsFv with a cleavable linker ............................................................................................... 20
2.5.3 Bispecific digoxigenin-binding antibodies ................................................................................. 21
2.6 FACS based cell surface receptor quantitation ................................................................................. 21
2.7 Screening of Tetraspanins as promising tumor-relevant targets ....................................................... 25
2.8 Biology of Tetraspanins and role in tumor progression .................................................................... 28
2.8.1 General features of Tspans ......................................................................................................... 28
2.8.2 How can Tspans interact with so many proteins? Tspan enriched microdomains (TEMs) ....... 30
2.8.3 TEMs are regulated by lipids and palmitoylation of Tspans ...................................................... 31
2.8.4 Integrins represent the main Tspan interaction partner .............................................................. 31
2.9 Tumor progression promoting Tspans............................................................................................... 32
2.9.1 Tumor progressing Tetraspanins: Tspan24 ................................................................................ 33
2.9.2 mAbs against Tspan24 ............................................................................................................... 35
2.9.3 Tumor progressing Tspans: Tspan8 (CO-029) ........................................................................... 38
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3 Aim of studies ......................................................................................................................................... 41
3.1 FACS-based Receptor Quantitation .................................................................................................. 41
3.2 Tetraspanin Screen ............................................................................................................................ 41

4 Materials ................................................................................................................................................. 43
4.1 Chemicals, enzymes and materials .................................................................................................... 43
4.2 Cell culture reagents .......................................................................................................................... 44
4.3 Receptor quantitation reagents & material ........................................................................................ 45
4.4 Antibodies ......................................................................................................................................... 45
4.4.1 Antibodies for Receptor quantitation and FACS ........................................................................ 45
4.4.2 Immunoblot antibodies ............................................................................................................... 46
4.5 siRNA components............................................................................................................................ 46
4.6 Lysis buffers and other buffers .......................................................................................................... 53
4.7 Commercial available Kits ................................................................................................................ 53

5 Methods ................................................................................................................................................... 55
5.1 Basic cell culture ............................................................................................................................... 55
5.2 FACS-based receptor quantitation .................................................................................................... 57
5.2.1 General procedure of FACS-based receptor quantitation ........................................................... 57
5.2.2 MESF reference standard and MESF calibration beads ............................................................. 58
5.2.3 Simple cellular beads (SCB) ...................................................................................................... 59
5.2.4 Receptor quantitation with QuantiBRITE .................................................................................. 60
5.2.5 Labeling of antibodies with Cy5 ................................................................................................ 60
5.2.6 mRNA expression profiling ....................................................................................................... 60
5.2.7 Quantitative confocal immunofluorescence analysis of bispecific antibodies ........................... 61
5.2.8 Antibody construction, expression and purification ................................................................... 61
5.3 Protein analysis ................................................................................................................................. 62
5.3.1 Whole cell extract preparation from eukaryotic cells ................................................................. 62
5.3.2 SDS-PAGE and Immunoblot ..................................................................................................... 63
5.3.3 BCA protein assay ...................................................................................................................... 64
5.3.4 Protein Expression and Purification - Transfection .................................................................... 64
5.3.5 Protein Expression and Purification - Protein Quantification .................................................... 65
5.3.6 Protein Expression and Purification – Immuno-precipitation .................................................... 65
5.3.7 Protein analysis - Reducing and Non Reducing SDS PAGE ..................................................... 65
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