Engineered Antibodies for the Therapy of Cancer and Inflammatory

Transcrição

Engineered Antibodies for
the Therapy of Cancer and
Inflammatory Diseases
Von der Fakultät Energie-, Verfahrens- und Biotechnik
der Universität Stuttgart zur Erlangung der Würde eines
Doktors der Naturwissenschaften (Dr. rer. nat.)
genehmigte Abhandlung
Vorgelegt von
Kirstin Anja Zettlitz
aus Wiesbaden
Hauptberichter: Prof. Dr. Roland Kontermann
Mitberichter: Prof. Dr. Klaus Pfizenmaier
Tag der mündlichen Prüfung:
27.09.2010
Institut für Zellbiologie und Immunologie
Universität Stuttgart
2010
I hereby declare that I performed the present thesis independently without further help or other
materials than stated.
Kirstin Anja Zettlitz
Stuttgart, July 20, 2010
Engineered Antibodies for the Therapy of Cancer and Inflammatory Diseases
Kirstin A. Zettlitz
Contents
Table of Contents
Table of Contents .................................................................................................................................... 3
Abbreviations .......................................................................................................................................... 6
Summary ................................................................................................................................................. 8
Zusammenfassung................................................................................................................................. 10
1 Introduction ................................................................................................................................... 12
1.1
Therapeutic antibodies.......................................................................................................... 12
1.2
Antibody structure ................................................................................................................ 17
1.3
Antibody functions ................................................................................................................ 18
1.3.1
Complement-dependent cytotoxicity, CDC ................................................................... 18
1.3.2
Antibody-dependent cellular cytotoxicity, ADCC .......................................................... 19
1.3.3
Cross-presentation ........................................................................................................ 19
1.3.4
FcRn ............................................................................................................................... 20
1.4
Antibody optimization ........................................................................................................... 20
1.4.1
Strategies to optimize structures: ................................................................................. 20
1.4.2
Immunogenicity ............................................................................................................. 21
1.4.3
Antigen binding ............................................................................................................. 21
1.4.3.1
Specificity............................................................................................................... 21
1.4.3.2
Affinity ................................................................................................................... 22
1.4.4
Fc domain modulation................................................................................................... 23
1.4.5
Pharmacological properties........................................................................................... 24
1.5
Targets ................................................................................................................................... 25
1.5.1
Heat shock protein 70 (Hsp70) in cancer ...................................................................... 25
1.5.2
Tumor necrosis factor (TNF) in inflammatory diseases................................................. 27
2 Materials and Methods ................................................................................................................. 30
2.1
Materials................................................................................................................................ 30
2.1.1
Instruments ................................................................................................................... 30
2.1.2
Special Implements ....................................................................................................... 30
2.1.3
Chemicals....................................................................................................................... 31
2.1.4
Peptides ......................................................................................................................... 31
2.1.5
Media and supplements ................................................................................................ 31
2.1.5.1
Bacterial culture .................................................................................................... 31
2.1.5.2
Cell Culture ............................................................................................................ 32
2.1.5.3
Antibiotics .............................................................................................................. 32
2.1.6
Solutions ........................................................................................................................ 32
2.1.7
Bacteria and phage ........................................................................................................ 33
2.1.8
Cell lines......................................................................................................................... 33
2.1.9
Antibodies...................................................................................................................... 33
2.1.10 Antigens ......................................................................................................................... 34
2.1.11 Enzymes ......................................................................................................................... 34
2.1.12 Restriction Enzymes ...................................................................................................... 34
2.1.13 Kits, Marker ................................................................................................................... 35
2.1.14 Primer ............................................................................................................................ 35
2.1.14.1
Primer for cloning of IgG humex ........................................................................... 35
2.1.14.2
Primer for Hsp70 fragments .................................................................................. 35
2.1.14.3
Primer for mutagenesis ......................................................................................... 36
2.1.14.4
Primer for screening or sequencing ...................................................................... 36
2.1.15 Vectors........................................................................................................................... 37
2.2
Methods ................................................................................................................................ 37
2.2.1
Cloning ........................................................................................................................... 37
3
Kirstin A. Zettlitz
Contents
2.2.1.1
Bacterial culture .................................................................................................... 37
2.2.1.2
Chemical competent E. coli cells ........................................................................... 37
2.2.1.3
Electro competent E. coli cells............................................................................... 37
2.2.1.4
Polymerase chain reaction .................................................................................... 38
2.2.1.5
Site directed mutagenesis ..................................................................................... 38
2.2.1.6
Restriction digest, Ligation, Ethanol precipitation ................................................ 39
2.2.1.7
Antibody humanization ......................................................................................... 39
2.2.1.8
Cloning of IgG humex ............................................................................................ 40
2.2.1.9
Cloning of Hsp70 fragments .................................................................................. 40
2.2.1.10
Cloning of chimeric TNFR1-Fc fusion proteins ...................................................... 41
2.2.1.11
Heat shock transformation of E. coli TG1 .............................................................. 41
2.2.1.12
Electroporation of E.coli TG1 ................................................................................. 41
2.2.1.13
Screening of clones ................................................................................................ 41
2.2.1.14
Plasmid-DNA Isolation (Midi, Mini) ....................................................................... 42
2.2.1.15
Sequence Analysis ................................................................................................. 42
2.2.2
Phage display ................................................................................................................. 42
2.2.2.1
Preparation of helper phage ................................................................................. 42
2.2.2.2
Immunotube selection .......................................................................................... 42
2.2.2.3
Equilibrium selection ............................................................................................. 43
2.2.2.4
Rescue of phage libraries for selection with helper phage ................................... 44
2.2.2.5
Polyclonal phage ELISA .......................................................................................... 44
2.2.2.6
Screening of soluble scFv by ELISA ........................................................................ 44
2.2.3
Coupling peptides to carrier proteins ........................................................................... 45
2.2.4
Biotinylation of antigen ................................................................................................. 45
2.2.5
Protein production ........................................................................................................ 45
2.2.5.1
Periplasmic protein expression in E. coli TG1 (scFv, Hsp70-SBD, AlF-fusion)........ 45
2.2.5.2
Protein expression in BL21-DE3 (Hsp70-fragments) ............................................. 46
2.2.5.3
Production of IgG humex in CHO-K1 ..................................................................... 46
2.2.5.4
Production of chimeric TNFR1-Fc in HEK293......................................................... 46
2.2.6
Protein purification........................................................................................................ 46
2.2.6.1
Immobilized Ni+ affinity chromatography, IMAC .................................................. 46
2.2.6.2
Protein A affinity chromatography ........................................................................ 47
2.2.7
Protein characterization ................................................................................................ 47
2.2.7.1
SDS-PAGE, Immunoblot and Western Blot analysis .............................................. 47
2.2.7.2
Thermal stability .................................................................................................... 48
2.2.7.3
Deglycosylation...................................................................................................... 48
2.2.8
Affinity measurements .................................................................................................. 48
2.2.9
ELISA .............................................................................................................................. 48
2.2.10 Flow cytometry .............................................................................................................. 49
2.2.11 Cytotoxicity .................................................................................................................... 49
2.2.12 IL-6 and IL-8 assays ........................................................................................................ 49
2.2.13 Preparation of peripheral blood mononuclear cells (PBMC) ........................................ 49
2.2.14 Isolation of human polymorphonuclear cells (PMN) and flow cytometry assay for
oxidative burst using dihydrorhodamine (DHR) ............................................................................ 50
3 Results ........................................................................................................................................... 51
3.1
Hsp70-specific antibodies for cancer therapy ....................................................................... 51
3.1.1
Chimerization of cmHsp70.1 (IgG tumex) ..................................................................... 51
3.1.2
Humanization of cmHsp70.1 ......................................................................................... 53
3.1.2.1
Analysis of mouse variable regions ....................................................................... 53
3.1.2.2
Design of reshaped human antibody .................................................................... 54
3.1.2.3
ScFv humex ............................................................................................................ 56
4
Kirstin A. Zettlitz
4
5
6
7
8
Contents
3.1.2.4
IgG humex.............................................................................................................. 57
3.1.3
Antibody glycosylation .................................................................................................. 58
3.1.4
Affinity measurement.................................................................................................... 59
3.1.5
Epitope mapping of Hsp70-specific antibodies ............................................................. 60
3.2
Antagonistic TNFR1-specific antibodies for therapy of inflammatory diseases.................... 64
3.2.1
Antibody constructs derived from humanized H398 (IZI-06.1) ..................................... 64
3.2.1.1
IZI-06.1 scFv ........................................................................................................... 64
3.2.1.2
IZI-06.1 Fab-HSA .................................................................................................... 65
3.2.1.3
IZI-06.1 scFv-HSA ................................................................................................... 66
3.2.1.4
IZI-06.1 IgG (ATROSAB) .......................................................................................... 66
3.2.2
TNFR1-Fc fusion proteins .............................................................................................. 68
3.2.3
Antigen specificity (species- and receptor-specificity) .................................................. 68
3.2.4
Thermal stability ............................................................................................................ 70
3.2.5
Affinity measurements .................................................................................................. 71
3.2.5.1
IZI-06.1 monovalent antibody constructs ............................................................. 71
3.2.5.2
ATROSAB, H398 ..................................................................................................... 72
3.2.6
Pharmacokinetics .......................................................................................................... 73
3.2.7
Antagonistic activity of TNFR1-specific antibodies ....................................................... 74
3.2.8
Epitope mapping ........................................................................................................... 78
3.2.9
Optimization of IZI-06.1................................................................................................. 79
3.2.9.1
Library construction............................................................................................... 80
3.2.9.2
Immunotube selection of library 1 (CDRH1/CDRL2) ............................................. 81
3.2.9.3
Immunotube selection of library 2 (CDRH2/CDRL1) ............................................. 83
3.2.9.4
Immunotube selection of library 2a (CDRH2) ....................................................... 83
3.2.9.5
Equilibrium selection of library 2a (CDRH2) .......................................................... 84
3.2.9.6
Immunotube selection of library 2b (CDRL1) ........................................................ 85
3.2.9.7
Summary of selections of CDR H1, H2, L1, L2 ....................................................... 87
3.2.9.8
ScFv IZI-06.1_mutants and combined scFv ........................................................... 88
3.2.9.9
Binding and activity of scFv IZI-06.1 derivates ...................................................... 88
Discussion ...................................................................................................................................... 91
4.1
Hsp70-specific antibodies for cancer therapy ....................................................................... 91
4.1.1
Chimerization of cmHsp70.1 (IgG tumex) ..................................................................... 91
4.1.2
Humanization of cmHsp70.1 (IgG humex) .................................................................... 92
4.1.3
Epitope mapping of Hsp70-specific antibodies ............................................................. 94
4.2
TNFR1-specific antibodies for therapy of inflammatory diseases......................................... 96
4.2.1
Mono- and bivalent derivatives of humanized IZI-06.1 ................................................ 96
4.2.2
Optimization of IZI-06.1................................................................................................. 99
References ................................................................................................................................... 103
Sequences.................................................................................................................................... 115
6.1
human Hsp70 ...................................................................................................................... 115
6.2
human TNFR1-Fc ................................................................................................................. 116
6.3
mouse TNFR1....................................................................................................................... 117
6.4
rhesus TNFR1 ....................................................................................................................... 117
Acknowledgements ..................................................................................................................... 118
Curriculum Vitae .......................................................................................................................... 119
5
Kirstin A. Zettlitz
Abbreviations
Abbreviations
°C
µ
aa
Ab / mAb
ADCC
Ag
AlF3
AML
amp
APS
AS
bp
BSA
CD
CDC
CDR
CEA
CL
CLL
CRD
CTL
CTLA4
Da
DC
DHR
DMSO
DNA
dNTP
DTT
EC50
ECD
ECL
EDTA
EGFR
degree celsius
micro- (x10-6)
amino acid
(monoclonal) antibody
antibody-dependent cellular
cytotoxicity
antigen
domain 3 of HSA
acute myeloid leukaemia
ampicillin
ammonium persulfate
ankylosing spondylitis
base pairs
bovine serum albumin
surface antigen, cluster of
differentiation
complement dependent
cytotoxicity
complementarity
determining region
carcinoembryonic antigen
constant light chain domain
chronic lymphocytic
leukaemia
cysteine rich domain
cytotoxic T lymphocyte
cytotoxic T lymphocyte
antigen 4
Dalton, molecular weight
dendritic cell
dihydro-rhodamine
dimethyl sulfoxide
deoxyribonucleic acid
deoxyribonucleoside
dithiothreitol
half maximal effective
concentration
extracellular domain
enhanced
chemiluminescence
ethylenediaminetetraacetate
epidermal growth factor
ELISA
Fab
FBS / FCS
Fc
FcRn
Fd
FDA
FITC
FR
Fv
g
glc
GS
h
HACA
HAHA
HAMA
HC / H
HER2
HPLC
HRP
HSA
Hsc70
Hsp70 / - SBD
hu
IC50
Ig / IgG
IL
IMAC
receptor
enzyme linked
immunosorbent assay
fragment antigen binding
fetal bovine / calf serum
fragment crystallizable
neonatal Fc-receptor
portion of the heavy chain
that is included in a Fab
food and drug
administration
fluorescein-5-isothiocyanate
framework region
fragment variable
gram
glucose
glutamine synthetase
hour
human anti-chimeric
antibody response
human anti-human antibody
response
human anti-mouse antibody
response
heavy chain of an antibody
human epidermal growth
factor receptor 2
high performance liquid
chromatography
horse radish peroxidase
human serum albumin
constitutive heat shock
protein 70
heat shock protein 70 / substrate binding domain
human
half maximal inhibitory
concentration
immunoglobulin (G)
interleukin
immobilized metal affinity
chromatography
6
Kirstin A. Zettlitz
ITAM
ITIM
JIA
k
kan
kcps
KD
l
LC / L
Lib / L
M
m
mA
MAC
MCS
MEF
MHC
min
MMP
mo
MOA
MS
MSX
mTNF / sTNF
MW / Mr
MWCO
n
NF-κB
NHL
NK cells
OD
PAGE
PBMC
PBS
PCR
PE
PEG
immunoreceptor tyrosinebased activation motif
immunoreceptor tyrosinebased inhibitory motif
juvenile idiopathic arthritis
kilo (x103)
kanamycin
kilocycle per second
(kilohertz)
dissociation constant
liter
light chain of an antibody
library
molar [1M=1mol/l]
mili- (x10-3), meter
milliampere
membrane attack complex
multiple cloning site
mouse embryonic fibroblast
major histocompatibility
complex
minute
matrix metallo-proteinase
mouse
mechanism of action
multiple sclerosis
L-methionine sulphoximine
membrane / soluble TNF
molecular weight / relative
molecular mass
molecular weight cut-off
nano- (x10-9)
nuclear factor-kappa B
Non-Hodgkin’s lymphoma
natural killer cells
optical density
polyacrylamide
electrophoresis
peripheral blood
mononuclear cell
phosphate buffered saline
polymerase chain reaction
pycoerythrin
polyethylene glycol
PK
PLAD
PMA
QCM
R
r
RA
res Ssq
rhe
Rmax
rpm
RT
s
scFv
SDR
SEC
SLE
TAA
TACE
TACI
TMB
TNF / TNFR
TRADD
TRAF
Tris
UC
VEGF
VH, VL
wt
Abbreviations
pharmacokinetics
pre-ligand binding assembly
domain
phorbol 12-myristate 13acetate, phorbol-diester
quartz crystal microbalance
receptor
recombinant
rheumatoid arthritis
residual sum of squares
rhesus macaque
maximal response
rotations per minute
room temperature
second
single-chain fragment
variable
specificity-determining
residues
size exclusion
chromatography
systemic lupus
erythematosus
tumour associated antigen
TNF alpha converting
enzyme
transmembrane activator
and calcium-modulating
cyclophilin ligand interactor
tetra-methylbenzidine
tumor necrosis factor /
receptor
TNFR associated death
domain
TNFR associated factor
tris-(hydroxymethyl)-aminomethane
ulcerative colitis
vascular endothelial growth
factor
variable domains of the
heavy and light chain of an
antibody, respectively
wild type
7
Kirstin A. Zettlitz
Summary
Summary
Detailed knowledge of the antibody structure and function allows researchers to engineer antibodies
on a rational basis to design therapeutic antibodies dependent on the target antigen, therapeutic
strategy and clinical setting. The development of therapeutic antibodies is a rapidly growing field
with more than 30 antibodies approved over the past 25 years. Thereof, cancers as well as
autoimmune and inflammatory diseases are the main indications.
The purpose of the first part of this thesis was the cloning, chimerization and humanization of the
monoclonal antibody (mAb) cmHsp70.1. This mouse mAb is specific for heat shock protein 70
(Hsp70) and able to bind the plasma membrane bound form (mHsp70), associated with various
cancers including breast cancer, head-and-neck cancer, and acute myeloid leukemia. Humanization
of cmHsp70.1 by grafting the complementarity-determining regions onto homologous human
germline genes resulted in an antibody (humex) possessing a similar affinity (3 nM) as the parental
antibody and an improved production and thermal stability. Epitope mapping confirmed that the
parental, chimeric, and humanized antibodies recognize the same region including amino acids 473504 of the Hsp70 substrate binding domain (SBD). Hence, this humanized antibody provides a basis
for further development of a mHsp70-specific antibody therapy.
The purpose of the second part of this thesis was to evaluate an appropriate format and an
optimization strategy for a humanized antibody (IZI-06.1) specific for human tumor necrosis factor
receptor 1 (TNFR1). Selective inhibition of TNFR1 provides the opportunity to neutralize the proinflammatory activity of TNF while maintaining the advantageous immunological responses mediated
by TNFR2. Here, this humanized antibody was converted into an IgG1 molecule (ATROSAB)
containing a modified human Fc region deficient in mediating effector functions. IgG ATROSAB was
compared to monovalent antibody derivatives fused to human serum albumin (HSA). Using chimeric
human/mouse TNFR1 molecules, the epitope of ATROSAB was mapped to the N-terminal region
(amino acid residues 1-70) comprising the first cysteine-rich domain (CRD1) and the A1 sub-domain
of CRD2. Purified ATROSAB, produced in CHO cells, inhibited in vitro typical TNF-mediated responses
like apoptosis induction and activation of NFκB-dependent gene expression such as IL-6 and IL-8
production. Moreover, ATROSAB showed strong binding to human and rhesus TNFR1-Fc fusion
protein with an affinity identical to the parental mouse antibody H398. These findings open the way
to further analyze the therapeutic activity of ATROSAB in relevant disease models in non-human
primates. Furthermore, phage display technology was used for affinity maturation of the humanized
variable domains by site directed mutagenesis of CDR1 and CDR2 of each VH and VL. Attempts
8
Kirstin A. Zettlitz
Summary
towards affinity maturation resulted in a mutant (scFv IG11) showing a two-fold increase in antigen
binding affinity which also translated into slightly improved inhibition of TNF-mediated cytotoxicity in
vitro.
The results of this study indicate that further engineering of ATROSAB could offer a number of
benefits for its therapeutic efficacy. TNFR1-selective antagonist, such as ATROSAB, will permit new
therapeutic options for diseases where anti-TNF therapeutics failed or even exacerbate disease
progression and could be an especially useful therapeutic alternative in diseases already known to
clinically respond to anti-TNF treatment and particularly in those diseases where specific blockage of
TNFR1 and maintenance of TNFR2 function appears as a promising therapeutic approach.
9
Kirstin A. Zettlitz
Zusammenfassung
Zusammenfassung
Detailliertes Wissen um die Struktur und Wirkungsweise von Antikörpern ermöglicht es
Wissenschaftlern, Antikörper nach rationalen Grundlagen zu gestalten, abhängig von Zielstrukturen,
therapeutischen Strategien und klinischen Vorgaben. Die Entwicklung von therapeutischen
Antikörpern ist ein schnell wachsendes Fachgebiet, mit über 30 zugelassenen Antikörpern in den
letzten 25 Jahren. Die häufigsten Indikationen davon sind Krebserkrankungen, sowie Autoimmunund entzündliche Erkrankungen.
Das Ziel des ersten Teils dieser Arbeit war die Klonierung, Chimärisierung und Humanisierung des
monoklonalen Antikörpers (mAb) cmHsp70.1. Dieser Maus-Antikörper ist spezifisch für das HitzeSchock-Protein 70 (HSP70) und bindet dessen Plasmamembran-gebundene Form, welche mit
verschiedenen Krebserkrankungen wie Brustkrebs, Kopf-Hals-Karzinom und akute myeloische
Leukämie assoziiert ist. Die Humanisierung, durch Transplantation der „complementaritydetermining regions“ (CDR) auf humane homologe Keimbahngene, resultierte in einem Antikörper
(humex) mit vergleichbarer Affinität (3 nM) zum ursprünglichen Antikörper und verbesserter
Produktivität und thermischer Stabilität. Die Epitop-Kartierung bestätigte, dass der parentale, der
chimäre und der humanisierte Antikörper dieselbe Region der Substrate-Binde-Domäne (SBD)
erkennen, diese Region umfasst die Aminosäuren 473-504. Folglich bietet dieser humanisierte
Antikörper eine Basis für die weitere Entwicklung einer Membran-Hsp70 spezifischen Antikörper
Therapie.
Die Zielsetzung im zweiten Teil dieser Arbeit war die Evaluierung eines geeigneten Formats und einer
Optimierungs-Strategie für einen humanisierten Antikörper (IZI-06.1), der spezifisch für den
humanen Tumor Nekrose Faktor Rezeptor 1 (TNFR1) ist. Die selektive Blockierung von TNFR1 erlaubt
die Neutralisierung der entzündlichen Wirkung von TNF, während die positiven TNFR2-vermittelten
Immunantworten bestehen bleiben. Dieser humanisierte Antikörper wurde hier in ein IgG Molekül
(ATROSAB) konvertiert, das einen modifizierten, Effektor-Funktion defizienten, humanen Fc Teil
enthält. IgG ATROSAB wurde mit monovalenten, Albumin-fusionierten (humanes Serum Albumin,
HSA) Antikörperfragmenten verglichen. Mittels chimärer (Human/Maus) TNFR1 Moleküle wurde das
Epitope von ATROSAB in der N-terminalen Region (Aminosäuren 1-70) kartiert, es umfasst die erste
CRD (Cysteine-reiche Domäne) und die Subdomäne A1 der CRD2. In CHO Zellen produzierter und
gereinigter IgG ATROSAB blockierte in vitro typische TNF-vermittelte Reaktionen wie die Induktion
von Apoptose und die Aktivierung von NFκB-abhängiger Genexpression, wie die Produktion von IL-6
und IL-8. Darüber hinaus zeigte ATROSAB starke Bindung an humanes und rhesus TNFR1-Fc
10
Kirstin A. Zettlitz
Zusammenfassung
Fusionsprotein mit einer identischen Affinität wie der parentale Maus-Antiköper H398. Diese
Ergebnisse ermöglichen die weitere Analyse der therapeutischen Wirkung von ATROSAB in
relevanten Krankheits-Modellen in nicht-humanen Primaten. Des Weiteren wurde zur AffinitätsReifung der humanisierten variablen Domänen Phagen-Display und gezielte Mutagenese (site
directed mutagenesis) der CDR1 und CDR2 von VH und VL eingesetzt. Die angestrebte AffinitätsReifung führte zu einer Mutante (scFv IG11) mit verbesserter Antigen-Bindungs Affinität, was zu
einer ebenfalls leicht verstärkten Blockierung von TNF-vermittelter Zytotoxizität in vitro führte.
Die Ergebnisse dieser Studie zeigen, dass weiteres Engineering von ATROSAB einige Vorteile für die
therapeutische Wirksamkeit bieten kann. TNFR1-spezifische Antagonisten, wie ATROSAB,
ermöglichen neue therapeutische Optionen für Krankheiten in denen Anti-TNF Therapeutika keine
Wirkung zeigten oder sogar den Krankheitsverlauf beschleunigten und könnten eine therapeutische
Alternative sein, für Krankheiten die klinisch auf anti-TNF-Behandlung ansprechen und besonders für
Krankheiten in denen die spezifische Blockierung von TNFR1 und die Erhaltung der TNFR2 Funktionen
ein vielversprechender therapeutischer Ansatz ist.
11
Kirstin A. Zettlitz
Introduction
1 Introduction
1.1 Therapeutic antibodies
The research and development of monoclonal antibodies is a rapidly progressing field (Aggarwal
2009; Beck et al. 2010) with more than 30 antibodies or antibody derivatives approved for human
therapy over the past 25 years (Beck et al. 2008; Reichert 2010). Thereof, cancer (Weiner et al. 2010),
and autoimmune and inflammatory diseases (Chan and Carter 2010), are the main indications. The
antibody properties of target specificity, low toxicity and the ability to activate the immune system
suggest the continuing promise of therapeutic antibodies.
The concept of using antibodies to selectively target tumors was proposed by Paul Ehrlich (“magic
bullet”-concept) over a century ago (Ehrlich 1906). However, only with the invention of the
hybridoma technology in 1975 (Köhler and Milstein 1975), the production of monoclonal antibodies
(mAb) became possible and led to the approval of the first mouse monoclonal antibody muromonab
eleven years later (1986, CD3-specific, Orthoclone OKT3; Janssen-Cilag) (Table 1-1). The mouse origin
of mAbs accounts for their therapeutic limitations: these antibodies are generally highly
immunogenic in humans, unable to induce human immune effector functions and have a short
serum half-life of typically less than 20 hours (Carter 2001; Presta 2002), because they do not bind to
the human neonatal Fc receptor (FcRn) (Ober et al. 2001). There are only two further mouse mAbs in
the clinic (ibritumomab tiuxetan, Zevalin; Biogen Idec Inc. and
131
I-toditumomab, Bexxar;
GlaxoSmithKline), both antibodies induce cell death by radiation and are approved for treatment of
non-Hodgkin’s lymphoma.
The event of antibody chimerization was first described in 1984 by S.L. Morrison (Morrison et al.
1984) (Table 1-1), and overcomes some of these limitations. A chimeric antibody is encoded by genes
from more than one species; usually the variable (V; antigen binding) domains of a mouse
monoclonal antibody are joined to the constant domains of a human antibody. The reduction of the
mouse portion of an antibody lowers the risk of a mouse-specific antibody response in humans
(HAMA, human anti-mouse antibodies), and enables the induction of Fc-mediated effector functions.
Abciximab (glycoprotein IIβ-specific, ReoPro; Centocor/Eli Lilly), the first chimeric Fab fragment was
approved in 1994, 10 years after the description of chimerization. The first full length chimeric
antibody
was
approved
in
1997
(rituximab,
CD20-specific,
Rituxan/Mabthera;
Genentech/Roche/Biogen Idec), followed by infliximab in 1998 (TNF-specific, Remicade;
Centocor/Merck). Even though chimeric antibodies show reduced immunogenicity, the immune
system can develop a human anti-chimeric antibody response (HACA) thus limiting the half-life and
12
Kirstin A. Zettlitz
Introduction
clinical efficacy of the therapeutic antibody (Hanauer et al. 2002; Baert et al. 2003) and can even lead
to anaphylactic reactions in some patients (Vultaggio et al. 2009).
In 1986 P.T. Jones et al. described antibody humanization as the next step to further increase the
similarity of the therapeutic antibody to human antibodies (Jones et al. 1986). A common
humanization method is CDR-grafting in which all the six complementarity determining regions
(CDRs; that is the antigen-binding loops) of the non-human antibody are grafted onto a framework
acceptor sequence of a human germline V gene that is similar to the variable domains of the
antibody of interest (Jones et al. 1986; Verhoeyen et al. 1988). It took eleven years until the first
humanized antibody was approved for immunosuppressive treatment in organ transplantations
(1997, daclizumab, CD25-specific, Zenapax; Hoffman-La Roche) and from the currently approved
therapeutic antibodies twelve are humanized.
A further milestone of antibody engineering was described in 1990 by J. McCafferty: the phage
display technology for the generation of fully human antibodies (McCafferty et al. 1990). This
technology is based on the physical linkage of genotype and phenotype of an antibody fragment by
displaying the protein on the surface of a bacteriophage that contains the gene encoding the
displayed protein. A fully human antibody derived from phage display (adalimumab, TNF-specific,
Humira; Abbott) was approved for the first time in 2002, 12 years after the description of phage
display. Another technology for the generation of fully human antibodies was introduced in 1994 by
L.L. Green (Green et al. 1994) (Abgenix mice) and by N. Lonberg (Lonberg et al. 1994) (Medarex
mice): genetically engineered (transgenic) mice that express human antibody repertoires. Human
antibodies are generated by antigen immunization to stimulate an in vivo humoral immune response
to generate somatically mutated, affinity matured, secondary repertoire B cells. Subsequently, the B
cells can be cloned and screened by conventional hybridoma technology. Those mAbs show often a
high binding affinity and specificity, reflecting the importance of combinatorial diversity (encoded in
the germline library of V, D, and J gene segments) and junctional and somatic diversity. At least 66
different therapeutic drugs, derived from either phage display or transgenic mice (e.g. 2006,
panitumumab, EGFR-specific, Vectibix; Amgen), have entered human clinical trials. The reported
binding affinities for these MAbs range over five orders of magnitude; however, most are
subnanomolar and nearly 50 % have affinities in the 0.1 - 1 nanomolar range, with no preference of
either technology. Yet, lead optimization as reported for phage display derived antibodies may not
be required for transgenic mouse derived antibodies that have already undergone affinity
maturation in vivo (Lonberg 2005).
13
Kirstin A. Zettlitz
Introduction
So far, second generation antibodies have entered the clinic. These follow-up antibodies originate
from approved therapeutic antibodies or are directed against the same validated antigens, but have
alterations such as improved variable domains to decrease immunogenicity (humanized or human
variable domains), altered affinity (affinity matured CDRs) or have different antibody formats (Fab
fragments or Fc-fusion proteins). Examples are certolizumab pegol (2008, TNF-specific, Cimzia; UCB),
a PEGylated Fab fragment of the IgG4 isotype, and golimumab (2009, TNF-specific, Simponi;
Centocor), a fully human antibody derived from transgenic mice, following infliximab.
In addition, third-generation antibodies have reached clinical trials (Strohl 2009; Oflazoglu and
Audoly 2010). These antibodies target different epitopes, trigger other mechanisms of action (MOA)
and are often engineered (glyco- or amino acid engineered Fc region) for improved Fc-associated
immune functions or extended half-life. For example, obinutuzumab (CD20-specific, GA101; Biogen
idec/Roche/Glycart) follows rituximab and is less immunogenic, has a different mechanism of action
and is glyco-engineered for increased effector functions (Oflazoglu and Audoly 2010).
Table 1-1: Therapeutic monoclonal antibodies and Fc fusion proteins for use in oncology, autoimmunity and
inflammation. Adapted from (Chan and Carter 2010).
year
generic name (trade
name; sponsoring
company)
1975
Production of monoclonal antibodies by hybridoma technology invented by C. Milstein and G. Köhler
(Köhler and Milstein 1975).
Antibody chimerization first described by S.L. Morrison et al. (Morrison et al. 1984).
muromonab-CD3
mouse
CD3ε
organ transplant
modulates T cell function
(Orthoclone OKT3;
IgG2a
rejection
Janssen-Cilag)
Antibody humanization first described by P.T. Jones et al. (Jones et al. 1986).
Description of phage display technology for the generation of fully human antibodies by J. McCafferty
and colleagues (McCafferty et al. 1990).
abciximab
chimeric
CD41,
cardiovascular
prevents blood clotting
(ReoPro; Centocor/Eli Fab (IgG1)
glycodisease
Lilly)
protein
IIb/IIIa
Description of transgenic mice with human immunoglobulin genes by L.L. Green et al. (Abgenix mice)
(Green et al. 1994) and by N. Lonberg et al. (Medarex mice) (Lonberg et al. 1994).
rituximab
chimeric
CD20
NHL, RA
sensitizes cells to chemo(Rituxan/Mabthera;
IgG1
(inadequate
therapy; induces apoptosis,
Genentech/Roche/
responses to TNF
ADCC and CDC
Biogen Idec)
blockade) and CLL
daclizumab
humanized CD25 (α
organ transplant
(Zenapax; HoffmannIgG1
chain of IL- rejection
La Roche)
2 receptor)
basiliximab
chimeric
CD25
organ transplant
(Simulect; Novartis)
IgG1
rejection
1984
1986
1990
1994
1997
1998
antibody
format
target
indication
proposed mode of action
14
Kirstin A. Zettlitz
Introduction
infliximab
(Remicade;
Centocor/Merck)
chimeric
IgG1
TNF
etanercept
(Enbrel;
Amgen/Pfizer)
trastuzumab
(Herceptin;
Genetech/Roche)
TNFR2
ECD-Fc
(IgG1)
humanized
IgG1
TNF
HER2
Crohn’s disease,
RA, psoriatic
arthiritis, UC, AS
and plaque
psoriasis
RA, JIA, psoriatic
arthritis, AS and
plaque psoriasis
breast cancer
2000
gemtuzumab
ozogamicin
(Mylotarg; Pfizer)
CD33
AML
2001
alemtuzumab
(Campath/MabCampa
th; Genzyme/Bayer)
111
90
In/ Y-ibritumomab
tiuxetan
(Zevalin; Biogen Idec)
adalimumab
(Humira/Trudexa;
Abbott)
humanized
IgG4calicheamicin
conjugate
humanized
IgG1
CD52
CLL, MS
ADCC
mouse
IgG2 radioconjugate
human
IgG1
(phage)
CD20
lymphoma
B cell depletion by radiation
TNF
RA, JIA, psoriatic
arthritis, Crohn’s
disease, AS and
plaque psoriasis
I-tositumomab
(Bexxar;
GlaxoSmithKline)
omalizumab
(Xolair; Genentech/
Roche/ Novartis)
mouse
IgG2 radioconjugate
humanized
IgG1
CD20
lymphoma
neutralizes TNF activity by
binding soluble and
transmembrane TNF; lyses
TNF-expressing cells by CDC;
induction of activated T cell
and macrophage apoptosis
B cell depletion by radiation
IgE-Fc
moderate to
severe persistent
allergic asthma
efalizumab
(Raptiva; Genentech/
Merck Serono)
alefacept
(Amevive; Astellas)
humanized
IgG1
CD11a
(LFA-1)
psoriasis
LFA3 ECDFc (IgG1)
CD2
SLE
cetuximab (Erbitux;
ImClone Systems/
Bristol-Myers-Squibb)
bevacizumab
(Avastin; Genentech)
chimeric
IgG1
EGFR
colorectal cancer
humanized
IgG1
VEGFA
colorectal, breast
and lung cancer
2002
2003
2004
131
binding soluble and
transmembrane TNF;
induction of activated T cell
and macrophage apoptosis
binding soluble and
transmembrane TNF
inhibition of receptor
dimerization; downregulation
of receptor; reduced
proliferation, angiogenesis
depletion of CD33 positive
blasts
ligand binding and receptor
antagonism, reduces release
of allergic response
mediators
inhibition of T cell migration
ligand binding and
neutralization; blocks
activation of TACI
inhibition of ligand binding
and dimerization of EGFR,
blocks EGFR activation; ADCC
blocks binding of VEGF to its
receptor; reduced
angiogenesis
15
Kirstin A. Zettlitz
2005
2006
natalizumab
(Tysabri;
Biogen Idec/Elan)
humanized
IgG4
α4 subunit
of
integrins
MS and Crohn’s
disease
nimotuzumab
(Theracim/Theraloc;
YM Biosciences/
Onco-sciences)
abatacept
(Orencia; BristolMyers Squibb)
humanized
IgG1
EGFR
squamous cell
carcinoma, head
and neck cancer
CTLA4
ECD-Fc,
mutated
IgG1-Fc
humanized
IgG1
CD80/
CD86
RA and JIA, UC
and Crohn’s
disease, SLE
IL-6R
RA, JIA
affinity
matured
Fab,
humanized
IgG1
human
IgG2
(mice)
humanized
IgG2-IgG4
hybrid
humanized
PEGylated
IgG4 Fab
human
IgG1
(mice)
mouse-rat
hybrid
VEGFA
age-related
macular
degeneration
anti-angiogenic, Inhibition of
all subtypes of VEGFA
EGFR
colorectal cancer
C5 complement
protein
TNF
Paraxysmal
nocturnal
haemoglobinuria
Crohn’s disease
and RA
TNF
RA, psoriatic
arthritis and AS
CD3 and
EPCAM
ovarian cancer,
malignant ascites,
gastric cancer
inhibition of ligand binding
and dimerization of EGFR,
blocks EGFR activation
binds C5, inhibiting its
cleavage and preventing the
formation of MAC
binding soluble and
transmembrane TNF
binding soluble and
transmembrane TNF
retargeting of T lymphocyte
to tumor cells; ADCC, CDC,
phagocytosis
human
IgG1
(mice)
human
IgG1
(mice)
IL-12 and
IL-23
plaque psoriasis
ligand binding and receptor
antagonism
CD20
CLL, RA
CDC and ADCC
tocilizumab
(Actemra/RoActemra;
Chugai/ Roche)
ranibizumab
(Lucentis; Genentech/
Novartis)
panitumumab
(Vectibix; Amgen)
2007
2008
2009
Introduction
eculizumab
(Soliris; Alexion
pharmaceuticals)
certolizumab pegol
(Cimzia; UCB)
golimumab
(Simponi; Centocor)
catumaxomab
(Removab; Fresenius
Biotech/Trion
Pharma)
ustekinumab
(Stelara; Centocor)
ofatumumab
(Arzerra; Genmab/
Glaxo-SmithKline)
receptor binding and
antagonism; inhibits
leukocyte adhesion to their
counter receptor
blocking of EGFR, reduced
proliferation
inhibits T cell activation by
binding to CD80 and CD86,
thereby blocking interaction
with CD28
receptor binding and ligand
blockade
The suffix of the international non-proprietary names for monoclonal antibodies denotes the antibody format: -omab,
mouse IgG2; -ximab, mouse–human chimeric IgG1; -zumab, humanized IgG1; -umab, human antibodies from phage
display or transgenic mice; -cept, Fc-fusion protein; -axomab, trifunctional (bispecific) mouse–rat hybrid.
16
Kirstin A. Zettlitz
Introduction
VL
Fab
Antigen
VH
CH 1
CL
Hinge
FcγR
Complement
CH 2
Fc
FcRn
CH 3
Sialyc acid
Glycosylation
Galactose
Bisecting
N-acetylglucosamine
Mannose
Core
Variable
Fucose
N-acetylglucosamine
Asn297
Figure 1-1: Antibody structure and function. IgGs comprise a pair of identical heavy and light chains. The heavy chain
contain a variable domain (VH) and three constant domains (CH1, CH2, CH3), whereas the light chain contain a variable
domain (VL) and a single constant domain (CL). IgGs contain two functional subunits: the fragment antigen binding (Fab)
and the fragment crystallizable (Fc). The Fc part interact with Fc receptors for IgG (FcγR), the complement component
C1q and the neonatal Fc receptor (FcRn). The Fc region is N-glycosylated at the conserved asparagines residue Asn297.
1.2 Antibody structure
Immunglobulin molecules are composed of two types of protein chains: heavy chains and light
chains. Two types of light chain, termed lambda (λ) and kappa (κ), are found in antibodies and do not
differ in function. Antibodies are grouped into five classes based on the sequence of their heavy
chain constant regions: IgM, IgD, IgG, IgE, and IgA. They differ in their biological properties,
functional locations and ability to deal with different antigens as depicted in Table 1-2. Thereof IgG is
the most commonly used for therapeutic antibodies (Reichert et al. 2005). IgG molecules are
tetramers of about 150 kDa, which comprise two identical heavy and light chains linked by disulphide
bonds. Antibodies can be divided into two distinct functional units: the fragment of antigen binding
(Fab) and the constant fragment Fc (fragment crystallizable; hinge and constant domains CH2 and
CH3). The Fab fragment is composed of one constant and one variable domain of each of the heavy
and light chain and contains the variable fragment (Fv), which is responsible for interaction with
17
Kirstin A. Zettlitz
Introduction
antigens. The antigen binding site is formed by six loops, the hypervariable complementarity
determining regions (CDRs), three of which are present in each of the VH and VL domains. The binding
site confers essential properties such as binding affinity and target specificity. The Fc fragment of an
antibody mediates its immune effector functions, by initiating complement-dependent cytotoxicity
(CDC), antibody-dependent cellular cytotoxicity (ADCC) and antibody-dependent cellular
phagocytosis (ADCP). A second function of the Fc is interaction with the neonatal Fc receptor (FcRn)
which controls the half-life of immunoglobulins.
Table 1-2: The structural and functional properties of the human immunoglobulin isotypes.
name types
IgA
α1, α2
description
antibody
half-life
complexes
in serum
mucosal areas, salvia, tears and breast milk; dimer
6 days
alternative pathway of complement activation; binding (Mr 390 kDa)
to macrophage and phagocyte Fc receptors
IgD
antigen receptor on B cells; activates basophils and monomer
mast cells
IgE
3 days
(Mr 184 kDa)
binds to allergens; triggers histamine release from monomer
2 days
mast cells and basophils; protects against parasitic (Mr 188 kDa)
worms; binding to macrophage and phagocyte Fc
receptors
IgG
γ1, γ2,
provides the majority of antibody-based immunity; monomer
γ3, γ4
FcRn
mediated
complement
placental
activation
(not
transport;
γ4);
21, 20,
classical (Mr 146 kDa; 7, 21
binding
to γ3: 165 kDa)
days
macrophage and phagocyte Fc receptors (not γ2)
IgM
expressed on the surface of B cells and in secreted pentamer
10 days
form with very high avidity; eliminates pathogens in (Mr 970 kDa)
the early humoral immunity, CDC
1.3 Antibody functions
1.3.1
Complement-dependent cytotoxicity, CDC
Subclasses of IgG (particularly IgG1 and IgG3) are strong activators of the classical complement
pathway. Antigen-bound antibodies on the cell surface lead to high-affinity binding of the
complement component 1q (C1q) to the Fc region and to subsequent activation of downstream
complement proteins. The result of the complement pathway is the formation of the membrane
18
Kirstin A. Zettlitz
Introduction
attack complex (MAC) and cell lysis. Moreover, immune effector cells (macrophages, mast cells and
granulocytes) are recruited and activated by the highly chemotactic complement molecules C3a and
C5a (Dunkelberger and Song 2010), bound to the target cell surface. Additionally, C3b acts as
opsonin, by binding to antigens and coating negatively charged molecules on the cell membrane,
thereby binding of phagocytic cells expressing complement receptor 1 (CR1) is greatly enhanced. The
relationship between complement activation and therapeutic activity is suggested from studies with
several clinically approved mAbs (Di Gaetano et al. 2003; Cragg and Glennie 2004; Racila et al. 2008).
1.3.2
Antibody-dependent cellular cytotoxicity, ADCC
ADCC is a mechanism of cell-mediated immunity whereby effector cells of the immune system that
express FcγR (mainly natural killer (NK) cells, but also neutrophils, mononuclear phagocytes and
dendritic cells (DCs)) actively lyse a target cell that has been bound by specific antibodies. FcγRs
mediate activating signals through immunoreceptor tyrosine-based activation motifs (ITAMs) and
inhibitory signals through immunoreceptor tyrosine-based inhibitory motifs (ITIMs). FcγRIIB (CD32) is
the major inhibitory FcR, whereas FcγRI (CD64) is a high affinity activating receptor expressed by
macrophages, DCs, neutrophils and eosinophils and FcγRIIIA (CD16A) is an activating receptor
expressed by NK cells, DCs, macrophages and mast cells (Nimmerjahn and Ravetch 2006).
The importance of Fc-FcγR interactions for the in vivo antitumor effects of therapeutic antibodies has
been shown in many studies (Clynes et al. 2000) and polymorphisms in genes encoding FcγRs are
associated with clinical response to antibodies (Cartron et al. 2002; Musolino et al. 2008; Bibeau et
al. 2009).
1.3.3
Cross-presentation
The lysis of tumor cells is a prerequisite for a process known as cross-presentation. Dendritic cells
(DCs) can engulf the resultant apoptotic tumor cells and subsequently present tumor-derived
peptides on MHC class I and II molecules (Berard et al. 2000). This dual presentation leads to direct
tumor cytotoxicity by CTLs (CD8+ cytotoxic T cells) (Albert, 1998) and promotes activation of CD4+ T
cells, which can prime B cells for the production of tumor-specific host antibodies.
The delayed therapeutic effect of e.g. rituximab points to a role of the adaptive immune system in
mediating long-term benefit of mAbs (Cartron et al. 2004). And there is increasing evidence to
suggest a role for cross-presentation in the induction of adaptive immune responses following
antibody therapy.
19
Kirstin A. Zettlitz
1.3.4
Introduction
FcRn
The neonatal Fc receptor (FcRn) is structurally distinct from FcRs and is related to MHC class I
molecules. FcRn plays an important role in passive transfer of humoral immunity from mother to
fetus. FcRn expressed on vascular endothelial cells bind to the Fc part of IgGs after fluid-phase uptake
at acidic pH in endosomes and return it to circulation, or protecting it from transcytotic lysosomal
catabolism en route to the lymphatics (Roopenian and Akilesh 2007). Thereby this recycling
mechanism contributes to the long half-life of IgG and maintenance of serum IgG. In addition, FcRn
may also contribute to antibody-mediated antigen presentation by dendritic cells by uniquely
directing multimeric immune complexes to lysosomes (Qiao et al. 2008).
1.4 Antibody optimization
Design goals for therapeutic antibodies are dependent on the target antigen, therapeutic strategy
and clinical setting. In anticancer therapy, antibodies are, as yet, rarely curative. Therefore, in
oncology the goal is to improve the antitumor activity of antibodies and improve patient survival, by
increasing the frequency of partial or complete responses and by extending the duration of response
(Carter 2001; Adams and Weiner 2005). In general, increasing the potency of the antibody or
extending its half-life in plasma might allow for a reduced dose or administration schedule, which
would reduce adverse effects and therapy costs and improve the patients’ quality of life.
Furthermore, it is often desirable to obtain species-crossreactive antibodies, allowing the biological
function of the antibodies to be evaluated in animal models of disease. However, it is challenging to
derive antibodies that are crossreactive with mouse antigens by immunization of mice or transgenic
mice, because self-reactive antibody-producing cells are selected against by the process of immune
tolerance induction.
Detailed knowledge of antibody structure and activity allows researchers to engineer primary
antibodies on a rational basis and even to introduce additional properties like increased cytotoxicity
or dual targeting or to generate IgG-related structures with additional functions and specificity.
1.4.1
Strategies to optimize structures:
Two broad strategies are widely used, either individually or in combination, for the optimization of
antibody therapeutics: display libraries (Lowe and Jermutus 2004; Hoogenboom 2005) and structurebased design (rational design) (Szymkowski 2005).
Phage display libraries are the most commonly used display technology for antibody generation and
optimization (Bradbury and Marks 2004; Lowe and Jermutus 2004; Hoogenboom 2005) but also
other display technologies, such as yeast-, mRNA-, and ribosome-display libraries are evolving
20
Kirstin A. Zettlitz
Introduction
(Hoogenboom 2005). A particular strength of display technology is that direct selection for specific
binding properties, such as higher affinity or species cross-reactivity, is sometimes possible (Popkov
et al. 2004). Display technology includes the selective recovery of antibodies on the base of antigen
binding, and provides the means to amplify the selected clone for further rounds of selection or
analysis. The binding step can be done with the antigen immobilized on a surface, in solution or on
cells and conditions can be adapted to the design goal (binding, stability, production). Experience
with phage display libraries supports the prediction that increases in functional display library size
(the number of clones that is displayed with sufficient efficiency to be potentially selectable)
facilitates the identification of higher-affinity clones (Perelson and Oster 1979; Griffiths et al. 1994;
Bradbury and Marks 2004).
Structure-based design uses three-dimensional structural information, sometimes in combination
with sophisticated computational methods, to predict the site and type of useful mutations
(Szymkowski 2005). The ideal starting point for design is a high-resolution structure of an antibody in
complex with the relevant binding partner (antigen, complement, FcRn, FcγR), but the molecular
model of an antibody can sometimes provide a useful starting point as well.
1.4.2
Immunogenicity
All protein drugs are potentially immunogenic (Chirino et al. 2004). Antibody chimerization
substantially reduces the immunogenicity of the antibody and humanization might further reduce it
(Hwang and Foote 2005). Beside the influence of the primary amino acid structure, other factors,
such as the presence of aggregated or misfolded antibody, contaminants, microheterogeneity in the
antibody pool (glycosylation, chemical modification) and the presence of T cell epitopes (Hwang and
Foote 2005) contribute to the immunogenicity of therapeutic antibodies.
1.4.3
Antigen binding
1.4.3.1 Specificity
Typically, antibody binding to its target antigen is of high specificity, which allows their use in
targeted therapy. On the other hand, it is less common for antibodies to bind the corresponding
antigen from a different species (e.g. non-human primate or rodent) although such crossreactivity
would enable preclinical evaluation of efficacy and toxicity. The sequence homology of the antigen in
different species (i.e. the conservation of the target epitope) is the crucial factor in generating such
species-crossreactive antibodies. Display technologies (phage display and others) are well suited to
the generation of species-crossreactive antibodies, because they are independent of the induction of
21
Kirstin A. Zettlitz
Introduction
immune tolerance in the immunization process and allow screening and/or selection for species
crossreactivity (Fredericks et al. 2004; Popkov et al. 2004).
1.4.3.2 Affinity
The antigen-binding affinity may be the most intensive studied property of antibodies, it describes
the ratio of the association (kon or ka) and dissociation (koff or kd) rate constants, which can be
measured using a range of technologies including quartz crystal microbalance (QCM) or surface
plasmon resonance (SPR). The affinity equilibrium constant (KD) is then calculated as a function of kon
and koff (KD = koff/kon). Affinity maturation to increase the strength of the antibody-antigen interaction
can be achieved using different library-construction strategies together with several display
technologies. Phage display has been successfully used to increase antibody affinity more than 1000fold (Yang et al. 1995; Schier et al. 1996c). In general, mutations are introduced into the antibody
gene, to create an antibody gene library. The key decision in this method is where and how to
introduce mutations into the antibody V-genes. The easiest approach is to introduce mutations
randomly into the V-genes, thus mimicking the in vivo process of hypermutation. Mutations can be
introduced by chain shuffling (Marks et al. 1992; Figini et al. 1994), error prone polymerase chain
reaction (PCR) (Hawkins et al. 1992), DNA shuffling (Crameri et al. 1996) or by reproduction of phage
in mutator strains of E. coli (Low et al. 1996). One limitation of random mutagenesis is that little
information is generated regarding to the localization of mutations that modulate affinity and the
constructed libraries are hardly large enough to cover the number of possible permutations. A
further problem is that this approach may create problems of immunogenicity. Another approach is
to introduce mutations site directed into the CDRs by using PCR and special randomized primers.
During in vivo somatic hypermutation, mutations accumulate preferentially in the CDRs compared to
framework residues (Loh et al. 1983). The location of these CDR mutations complements the location
where diversity is generated in primary antibody repertoire (Tomlinson et al. 1996). However, many
CDR residues, especially in VHCDR3 and VLCDR3, are responsible for high energy interactions with
antigen. Mutating these residues will in many cases abolish antigen binding, for that reason the
constructed library has to be large enough (according to the number of mutated residues) to allow
recreation of these important residues. Mutated residues in the CDRs may increase affinity by
introducing new contact residues or by replacing low affinity contact residues with more favorable
energetic (Novotny et al. 1989). Another possibility is that the introduced mutations indirectly
influence affinity by repositioning the CDRs or the side-chains of contact residues for optimal
interaction with the antigen (Foote and Winter 1992). Mutations in the CDRs are also less likely to
induce immunogenicity problems then mutations in the conserved framework regions. Finally, site
directed mutagenesis reveals information about conserved structural and functional residues and
22
Kirstin A. Zettlitz
Introduction
those residues that modulate affinity (Schier et al. 1996b) and these information will be useful for
subsequent mutagenic efforts. Separate mutations that increase binding affinity can be combined
and often further increase affinity in an additive or synergistic manner (Wells 1990).
Affinity maturation is more often than not (Carter et al. 1992) associated with increased in vitro
biological potency (Chowdhury and Wu 2005; Hoogenboom 2005). Not so much is known about the
relevance of affinity for the in vivo activity of an antibody. However, the antigen-binding affinity of an
antibody definitely influences its tumor localization, as shown for a panel of scFvs specific for ErbB2
(Adams et al. 1998; Adams et al. 2001). For specific tumor localization an affinity (KD) higher than
10-7 - 10-8 is needed and an affinity higher than 10-9 – 10-11 did not further improve tumor uptake. The
term “binding-site barrier” describes the observation that scFv with the lowest affinity had the most
uniform distribution throughout the tumor, while the highest affinity scFv was found mainly in the
perivascular region of the tumor (Fujimori et al. 1989). Interestingly, tumor localization of dimeric
versions (diabodies) of these ErbB2-specific scFvs correlated inversely with the monomeric affinity,
with the lowest-affinity diabody (KD of 400 nM) localizing more than twofold more efficiently than
the highest-affinity diabody (KD of 2 nM) (Nielsen et al. 2000). That hints to an influence of the
antibody format (valency, pharmacokinetics) which can outrun the influence of intrinsic affinity, as
long as tumor penetration is concerned. Another aspect is the nature and localization of the target
antigen, e.g. targets in inflammatory diseases are often soluble (like cytokines) or expressed on
immune cells, hence tissue penetration may not influence the biological potency of therapeutic
antibodies and the influence of the antigen-binding affinity may be different. The antigen-binding
affinity of current antibody therapeutics ranges from a KD of 0.08 nM (gemtuzumab ozogamicin,
Mylotarg; Wyeth) to 32 nM (alemtuzumab, Campath; Genzyme Corporation and Schering AG). If
there is an ideal antigen-binding affinity for antibodies remains unknown and an empirical approach
is warranted for preclinical optimization of antibody affinity.
1.4.4
Fc domain modulation
Mutational analysis has increased our understanding of, and provided a means to modulate, the
interaction of the Fc region of IgG with various FcγRs (Duncan et al. 1988; Shields et al. 2001), the
FcRn and complement component 1q (C1q) (Duncan and Winter 1988; Idusogie et al. 2000). Effector
functions mediated by the Fc region of an IgG might be increased with the intention to increase
efficacy or be decreased to reduce side-effects, according to the chosen target antigen, therapeutic
strategy and clinical setting.
For some antibody therapies (like inflammatory diseases), antigen binding may be sufficient for
achieving efficacy, and effector functions may be unnecessary and a potential source of adverse
23
Kirstin A. Zettlitz
Introduction
events in patients. There are different means to minimize FcγR binding, like the use of the isotypes
IgG2 and IgG4. Substitution into human IgG1 of IgG2 residues at positions 233-236 and IgG4 residues
at positions 327, 330 and 331 greatly reduced ADCC and CDC (Armour et al. 1999; Shields et al.
2001); a similar IgG (hIgG1e3, InvivoGen, San Diego, USA) was used in this study to generate a TNFR1specific humanized antibody (ATROSAB). Another means is the use of IgG of various isotypes with
mutations that impair interaction with FcγRs or prevent the glycosylation that is required for effector
functions (Alegre et al. 1994; Salfeld 2007; Labrijn et al. 2008) e.g. by mutating the conserved
asparagine
residue
at
position
Asn297
in
the
CH2
domain
(otelixizumab,
TRX4;
Tolerx/GlaxoSmithKline) (Bolt et al. 1993). Finally, antibody fragments lacking the Fc region can be
used (certolizumab pegol) (Choy et al. 2002).
On the other hand, Fc-mediated effector functions like ADCC and CDC are suggested to be important
mechanisms of action for several antibodies used in cancer immunotherapy. The modification of Fcmediated activities is emerging as one of the most promising ways to further increase the clinical
potential of antibodies (Carter 2006; Presta 2008; Kubota et al. 2009). One such Fc mutant
(Ser239Asp, Glu330Leu, Ile332Glu) gave a ~100-fold increase in ADCC potency as shown with variants
of several therapeutically relevant antibodies (Lazar et al. 2006). Increasing complement activity can
be achieved by isotype chimerism, if some IgG1 residues are replaced with IgG3 sequences (Natsume
et al. 2008) or by mutations to improve C1q binding, which showed ~two-fold greater CDC activity
but with slightly impaired ADCC activity (Idusogie et al. 2001; Coiffier et al. 2008).
Modification of the oligosaccharide content of the Fc domain provides another mechanism for
regulation of anti- and proinflammatory properties of therapeutic antibodies (Jefferis 2009b;
Jefferis 2009a), e.g. defucosylation leads to a significant enhancement of ADCC (Kubota et al. 2009).
The relative importance of each mechanism, and whether these mechanisms are synergistic, additive
or antagonistic, remains uncertain.
1.4.5
Pharmacological properties
The terminal half-life of antibodies can be adjusted over a wide range to fit clinical goals by tailoring
the interaction between IgG and FcRn. Mapping of the FcRn interaction site on IgG revealed two
conserved histidine residues (His310, His435) which are responsible for the pH dependence of this
interaction (Ghetie and Ward 2002).
Most approved antibodies (chimeric, humanized, human) are IgG1 with similar constant domains,
but their half-life vary from ~3 – 27 days (Lobo et al. 2004). Increased serum half-life and exposure
offers the potential benefits of greater efficacy, lower or less frequent dosing, lower cost and
24
Kirstin A. Zettlitz
Introduction
enhanced localization to the target. Different mutations of the Fc region resulted in increased
binding to the FcRn and extended serum half-life (Hinton et al. 2004; Dall'Acqua et al. 2006; Zalevsky
et al. 2010). The half-life of antibody fragment can also be extended by endowing them with the
ability to bind serum proteins as IgG or albumin; reviewed in (Kontermann 2009), or by conjugating
them to polyethylene glycol (PEGylation) (Choy et al. 2002).
1.5 Targets
A limitation of antibody therapeutics clearly is the restriction of targets to those on the cell surface or
exterior of host cells. The first step in the generation of antibody therapeutics, which is one of the
most difficult steps, is the selection of a target antigen; reviewed in (Carter et al. 2004). The selection
of antigen targets can be classified into two main approaches: the first one is the development of
antibodies against “validated targets”, i.e. prior mAbs have shown proof of activity and vast literature
exists on the importance of these targets for the disease mechanism. The second approach is the
“functional approach”, which intends to indentify new or less studied targets that confer particular
functions that might be involved in disease by selection of antibodies based on a functional screen.
Most cytokines and the associated receptors seem to be valuable targets for treatment of
immunological disorders as shown by the large number of already approved mAb (Strohl 2009; Chan
and Carter 2010; Smith and Clatworthy 2010). The mechanism driving at least some inflammatory
disorders are reasonably well known.
In contrast, finding new targets in oncology is a challenging issue (Weiner et al. 2010) because
malignancies have often multifactorial, redundant causes that are frequently poor understood. In
addition, patients may become resistant to current cancer treatment, leading to the expression of
new molecules (potential targets) that promote tumor growth (Spector and Blackwell 2009). Possible
tumor targets include antigens expressed by the tumor itself or in the tumor microenvironment (e.g.
growth factor receptors, increased during tumorgenesis).
1.5.1
Heat shock protein 70 (Hsp70) in cancer
Heat shock proteins (Hsp) belong to a large group of highly conserved proteins divided into several
families according to their molecular weight. Each family is composed by members expressed either
constitutively or regulated inducible and inhabit nearly all cellular compartments. They play a major
role in maintaining protein homeostasis and protecting the cell against stress-induced damage
(Lindquist and Craig 1988). Elevated levels of intracellular Hsps are found after environmental stress,
but also during differentiation, proliferation, and maturation. Members of the highly conserved
Hsp70 family, including the constitutively expressed Hsc70 and the stress-inducible Hsp70, are
25
Kirstin A. Zettlitz
Introduction
intrinsic to cellular life (Calderwood et al. 2005). They comprise a C-terminal region that binds
peptides and denatured proteins, and an N-terminal ATPase domain controlling opening and closing
of the substrate-binding domain (Mayer and Bukau 2005). Besides cytosolic occurrence, the inducible
Hsp70 is capable of translocating into the plasma membrane and can also be released into the
extracellular space in a membrane-associated form where it can exert immune stimulatory activities
(Sherman and Multhoff 2007; Vega et al. 2008). Membrane-associated Hsp70 (mHsp70) has been
selectively detected on more than 50 % of tumors of different origin, including lung cancer, colorectal
cancer, breast cancer, head-and-neck cancer, melanoma, and leukemia, but not on cells from normal
tissues (Multhoff et al. 1995; Hantschel et al. 2000; Farkas et al. 2003; Gehrmann et al. 2003;
Kleinjung et al. 2003; Steiner et al. 2006; Multhoff 2007) (Figure 1-2).
Figure 1-2: Hsp70 surface expression is tumor-specific and increased on metastasis. Table adapted from (Hantschel et al.
2000).
The translocation of Hsp70 into the plasma membrane of tumor cells makes it a promising target for
cancer therapy. Thus, mHsp70 can act as a recognition structure for natural killer (NK) cells and
antitumor activity was demonstrated, for example, with NK cells stimulated ex vivo with an Hsp70derived peptide (Multhoff et al. 1997; Multhoff et al. 2000; Multhoff et al. 2001; Multhoff 2002;
Krause et al. 2004; Multhoff 2009). A detailed analysis of various monoclonal antibodies recognizing
different epitopes revealed that parts of the C-terminal substrate-binding domain (SBD) of Hsp70 are
exposed on tumor cells (Botzler et al. 1998). One of these monoclonal antibodies, cmHsp70.1 (clone
C92F3B1), which was raised by immunizing mice with an SBD-derived synthetic peptide (Multhoff
2007), has been extensively studied and identified to be useful for diagnostic and therapeutic
applications (Moser et al. 2002; Gross et al. 2003; Gastpar et al. 2004; Gehrmann et al. 2004; Gastpar
et al. 2005; Pfister et al. 2007; Vega et al. 2008). Because mHsp70 has no proliferative activity, in vivo
anti-tumoral effects will be mediated through the Fc-part (ADCC, CDC) and the mouse antibody
cmHsp70.1 will be of limited use as therapeutic in humans.
26
Kirstin A. Zettlitz
Introduction
Conversion of a murine antibody into a chimeric or humanized derivative has been established as an
adequate option to reduce the murine portion of the antibody and to increase the therapeutic
efficacy (see 1.4.2, 1.4.4) (Almagro and Fransson 2008).
The purpose of the first part of this thesis was to clone and produce a recombinant chimeric antibody
derived from the mouse antibody cmHsp70.1 as well as the humanization of this antibody by CDRgrafting. Comparison of the recombinant antibodies and the parent mouse antibody regarding
production, stability, epitope specificity (species crossreactivity) and antigen-binding affinity should
show if these antibodies can be useful for further development of therapeutic antibodies and
antibody derivatives targeting the membrane-bound form of Hsp70.
1.5.2
Tumor necrosis factor (TNF) in inflammatory diseases
Tumor necrosis factor (TNF) is a pleiotropic cytokine and a central mediator of inflammation.
Elevated levels of TNF are associated with various inflammatory diseases including rheumatoid
arthritis, psoriasis, and Crohn's disease (Figure 1-3).
Figure 1-3: Schematic presentation of TNF action in rheumatoid arthritis. Adapted from (Pope 2002). An unknown trigger
sets up an initial inflammation in the synovial membrane that attracts autoreactive lymphocytes and macrophages to the
inflamed tissue. The produced cytokines (mainly IL-1 and TNF) stimulate monocytes, macrophages, endothelial cells and
fibroblasts and these cells produce more cytokines (IL-6, IL-8, MCP1, GM-CSF). Finally, this leads to the production of
matrix metallo proteinases, responsible for tissue destruction and to the activation of bone-destroying osteoclasts and
joint destruction.
Several TNF-neutralizing reagents have been approved for the treatment of these diseases, including
soluble TNF receptors (etanercept) as well as anti-TNF antibodies (infliximab, adalimumab,
certolizumab pegol, golimumab), and many more are under development; for review see: (Tracey et
al. 2008; Kontermann et al. 2009). The TNF antagonists are presently the most successful class of
27
Kirstin A. Zettlitz
Introduction
biological drug for inflammatory diseases (total worldwide sales of $16.4 billion in 2008 (Chan and
Carter 2010)) and with over 1 million patients treated with TNF antagonists, therapeutic efficacy is
well documented (Schiff et al. 2006). However, global TNF inhibition over a prolonged period of time
increases the risk of tuberculosis reactivation, serious infections and even malignancies (Bongartz et
al. 2006; Desai and Furst 2006; Wallis 2008). Consequently, medical information of all approved antiTNF medicines includes extensive warnings and precautions.
Figure 1-4: Strategies to interfere with TNF action.
Two TNF receptors (CD120a, TNFR1, CD120b, TNFR2) mediate signal transduction upon binding of
TNF (Locksley et al. 2001) (Figure 1-4). They consist of four extracellular cysteine-rich domains
(CRDs), a single CRD typically including six cysteine-residues distributed in a range of 40 amino acids.
They differ in their intracellular domains, with only TNFR1 containing a death domain (Wilson et al.
2009). TNFR1 is ubiquitously expressed on most cell types and is activated both by the membranebound form of TNF (mTNF) and soluble TNF (sTNF), which is produced from mTNF by proteolytic
cleavage (Black et al. 1997). TNFR1 is a strong activator of both gene expression and apoptosis and is
the major mediator of TNFs pathologic phenotypes. In contrast, TNFR2, expressed in a more
restricted manner e.g. by immune cells, endothelial cells and neurons, is mainly activated by mTNF.
Activation of TNFR2 induces anti-apoptotic signals via activation of the NF-κB pathway and can lead
to cell proliferation in vitro (Wajant et al. 2003). Furthermore, TNFR2 appears to play a role in
infection, tissue homeostasis and regeneration (Wallach and Kovalenko 2009) and can act in a
neuroprotective manner (Marchetti et al. 2004).
28
Kirstin A. Zettlitz
Introduction
All the currently approved TNF-targeting therapeutics interfere with TNF-action at the level of ligand
binding, thereby generally blocking TNF signaling affecting both receptors. Selective inhibition of
TNFR1 signaling has gained increasing attention as alternative to global TNF neutralization. Recently,
a TNF mutein (R1antTNF) selectively neutralizing the activity of TNFR1 has been described (Shibata et
al. 2008b). This TNF mutein, administered either as unmodified or as PEGylated protein (PEGR1antTNF), demonstrated therapeutic efficacy in acute murine hepatitis models and a murine
collagen-induced arthritis model (Shibata et al. 2008a; Shibata et al. 2009). The beneficial effect of
selectively inhibiting TNFR1 was further supported by results from a dominant-negative TNF mutein
(XPro1595), which is capable of forming inactive complexes with sTNF, thus selectively inhibiting the
pro-inflammatory action mediated by TNFR1 while preserving the innate immunity to infections
(Zalevsky et al. 2007; Olleros et al. 2009). TNFR1-selective inhibition can be also achieved with
TNFR1-specific antibodies. For example, a monoclonal antibody, H398, with selectivity for human
TNFR1, showed potent inhibition of TNF-mediated signal transduction and cytotoxicity (Thoma et al.
1990; Kruppa et al. 1992; Moosmayer et al. 1995).
Recently, a humanized version of H398 (Kontermann et al. 2008) was generated. This humanized
antibody (IZI-06.1), produced as Fab fragment in E. coli, exhibited in vitro neutralizing activities
comparable to that of the Fab fragment of the parental antibody. Hence, IZI-06.1 may open a new
treatment option by selectively inhibiting TNFR1-mediated signal transduction.
In the second part of this study, IZI-06.1 was converted into a full IgG1 antibody (ATROSAB). In order
to avoid Fc-mediated effector functions, an ADCC and CDC-deficient heavy chain was used (see
1.4.4). Additionally, monovalent IZI-06.1-derivatives (scFv, Fab, scFv-HSA, Fab-HSA) were used to
compare antigen-binding (affinity and specificity) and neutralizing behavior with the intention to find
the most appropriate antibody format for further development of a therapeutic TNFR1-specific
antibody drug. Furthermore, phage display technology was used for affinity maturation of the
humanized variable domains by site directed mutagenesis of CDR1 and CDR2 of each VH and VL.
29
Kirstin A. Zettlitz
Methods
2 Materials and Methods
2.1 Materials
2.1.1
Instruments
Balance
Blotter
Centrifuges
Electrophoresis
Electroporation
Film developing
machine
Flow cytometer
Gel documentation
Feinwaage Basic [Sartorius AG, Göttingen, Germany]
440-39N and 440-33N [Kern, Balingen, Germany]
Trans-Blot SD Semi-dry transfer cell [Bio-Rad, Munich, Germany]
Eppendorf 5804R [Eppendorf, Hamburg, Germany]
J2-MC, rotors: JA10, 14, 20 [Beckman Coulter, Krefeld, Germany]
Avanti-J301, rotors: JA10, 14, 30.5 [Beckman Coulter, Krefeld, Germany]
CR 422 [Jouan, Rennes, France]
GR4i [Jouan, Rennes, France]
Mini-PROTEAN®3 Cell Electrophoresis System [Bio-Rad, Munich, Germany]
Ready Agarose Precast Gel Electrophoresis System [Bio-Rad, Munich,
Germany]
Power Pac Basic and HC [Bio-Rad, Munich, Germany]
Gene-Pulser™, Pulse Controller, Capacitance Extender *Bio-Rad, Munich,
Germany]
Curix60 [Agfa, Cologne, Germany]
Cytomics FC 500 [Beckman Coulter, Krefeld, Germany]
Transilluminator, Gel documentation system Felix [Biostep, Jahnsdorf,
Germany]
Heat block
HBT-1-131 [Hlc–Haep Labor Consult, Bovenden, Germany]
HPLC system
Waters HPLC-System [Millipore, Billerica, USA]
Chromatography Software Clarify Lite v.2.4.1.65
Incubator for
BD 53 [Binder, Tuttlingen, Germany]
bacteria
Infors HAT Multitron 2 [Infors Ag, Basel, Switzerland]
Incubator for cell
Heal Force water-jacketed CO2 Incubator HF160W, [Shanghai Lishen, Shanghai,
culture
China]
Magnetic stirrer
MR 3001K 800W [Heidolph Instruments, Nürnberg, Germany]
Microplate reader
Microplate Reader Spectra max 340 PC [Molecular Devices, Palo Alto, USA]
Tecan infinite M200 [Tecan, Crailsheim, Germany]
PCR cycler
RoboCycler 96 [Stratagene, La Jolla, USA]
Pump
Ismatec mp13 GJ-10 [Ismatec, Wertheim-Mondfeld, G]
QCM
Attana A100 C-Fast System [Attana, Stockholm, Sweden]
Rotator
PTR-30 [Grant-bio, Cambridgeshire, UK]
Spectrophotometer Nanodrop® ND-1000 [pEQLab Biotechnology, Erlangen, Germany]
eppendorf BioPhotometer plus [eppendorf, Hamburg, Germany]
Vortex
Sky Line [Elmi Ltd., Riga, Latvia]
Water bath
MA6, [Lauda, Lauda-Königshofen, Germany]
Haake DC10, [Thermo Haake, Karlsruhe, G]
Zetasizer
Zetasizer Nano ZS, [Malvern Instruments, Herrenberg, Germany]
2.1.2
Special Implements
BioRad Ready Gel
Tris-HCl gel, 4-15 % linear gradient, cat.no. 161-1176 [Bio-Rad Laboratories,
Munich, Germany]
30
Kirstin A. Zettlitz
Cryobox
Dialysis tubes
Dynabeads
Electroporation
cuvettes
ELISA plates
HPLC column
IMAC affinity
matrix
Immunotubes
Liquid
Chromatography
Columns
magnetic
separator
Nitrocellulose
membrane
Protein ASepharose
Sensor chip
Dialysis Chamber
Square plates
Tissue culture
flasks and dishes
Whatman paper
X-ray films
2.1.3
Methods
filled with isopropanol for eukaryotic cell freezing [Nalgene, Rochester, USA]
ZelluTrans, 40 mm cut-off 8 – 10 kDa [Roth, Karlsruhe, Germany]
23 mm cut-off 12,4 kDa [Sigma-Aldrich, Taufkirchen, Germany]
Dynabeads®M-280 Streptavidin, Cat.no. 112.05D [Invitrogen Dynal, Carlsbad,
USA]
1-mm-electroporation cuvettes, no. 71-2010 (red) [pEQLab, Erlangen,
Germany]
MICROLON 96 well [Greiner bio-one, Kremsmünster, Austria]
Nunc MaxiSorp™ *Thermo Scientific Fisher, Langenselbold, Germany]
BioSep-SEC-S2000 or S3000 [Phenomenex, Aschaffenburg, Germany]
BioSuite™ 250, 5 µm HR SEC *Waters GmbH, Eschborn, Germany+
His-SelectTM Nickel Affinity Gel [Qiagen, Hilden, Germany]
5 ml immunotubes *Nunc MaxiSorp™, cat.no. 444202, Thermo Scientific
Fisher, Langenselbold, Germany]
Luer-Lock, Non-jacketed, Size: 1.0 cm x 10 cm, bed: 8 ml, [Sigma-Aldrich,
Taufkirchen, Germany]
MPC-S [Dynal-Invitrogen, Carlsbad, USA]
BioTrace NT Membrane [Pall Life Sciences, East Hills USA]
CL-4B, P-3391 [Sigma-Aldrich, Taufkirchen, Germany]
Biotin Nc MkIII, prod.no. 3613-3033; Carboxyl Nc MkIII, prod.no. 3616-3103
[Attana, Stockholm, Sweden]
Slide-A-Lyzer®Dialysis Cassettes: Prod.no 66380, 0.5 – 3 ml Capacity, Cutoff
(MWCO) 10,000 [Thermo Scientific, Rockford, USA]
24 x 24 cm Tissue culture plates [Nunc, Thermo Scientific Fisher,
Langenselbold, Germany]
Cellstar® [Greiner bio-one, Kremsmünster, Austria]
3 mm 46 x 57 cm #3030 917 [Schleicher & Schuell, Brentford, UK]
Medical X-Ray film 100 NIF 18 x 23 cm [Fuji, Düsseldorf, Germany]
Chemicals
All chemicals were purchased by Roth [Karlsruhe, Germany], Boehringer Mannheim [Mannhein,
Germany], Merck [Darmstadt, Germany], Sigma-Aldrich [Taufkirchen, Germany] and Roche, [Basel,
Switzerland] unless otherwise stated and were of p.a. quality.
2.1.4
NLL-17
RFE-13
TKD-14
2.1.5
Peptides
NLLGRFELSGIPPAPRG
RFELSGIPPAPRG
TKDNNLLGRFELSG
[EMC microcollections, Tübingen, Germany]
Media and supplements
2.1.5.1 Bacterial culture
IPTG
Isopropyl-β-D-thiogalactopyranoside, stock solution 1 M in H2O [Gerbu
31
Kirstin A. Zettlitz
Methods
Biochemicals, Gaiberg, Germany]
1 % peptone, 0.5 % yeast extract, 0.5 % NaCl (low salt = 5 g/L) in H2O
LB medium, 1.5 % (w/v) agar, 100 µg/ml ampicillin, 1 % glucose
0.5 % (w/v) yeast extract, 2 % (w/v) peptone, 10 mM NaCl, 2.5 mM KCl, 10
mM MgSO4, 10 mM MgCl2
SOB medium and 20 mM glucose (catabolite repression due to glucose)
1.6 % peptone, 1 % yeast extract, 0.5 % NaCl in H2O
2xTY medium and 1.5 % agar, 100 µg/ml ampicillin, 1 % glucose
10 %, 50 %, 86 % in H2O, 0.2 µm sterile filtered
0.5 M Na2HPO4, 0.22 M KH2PO4, 5 % NaCl, 10 % NH4Cl, pH 7.2
1.5 % agar, 0.2 % glucose, 1 mM MgSO4, 2 µg/ml vitamine B1, M9 stock 1x
0.7 % agar in 2xTY medium
LB medium, 1 x
LBamp, glc agar plates
SOB
SOC
TY medium, 2 x
TYEamp, glc
glycerol
M9 stock, 10 x
Minimal plates
TYE top agar
2.1.5.2 Cell Culture
DMSO
Eosin
FBS
FCS dialyzed
Lipofectamine™
2000
RPMI Media 1640
Opti-MEM
MSX
Trypsin/EDTA, 10 x
Lysis-buffer
99.5 % [Roth, Karlsruhe, Germany]
0.4 % Eosin, 10 % FBS, 0.02 % NaN3 in PBS
Heat inactivated at 56 °C for 1 hour, HyClone®, Cat.no. SV30160.03 [Thermo
Scientific, Cramlington, UK]
[GIBCO Invitrogen, Karlsruhe, Germany]
Cat.no. 11668-019 [Invitrogen, Karlsruhe, Germany]
+ L-glutamine and no glutamine [GIBCO Invitrogen, Karlsruhe, Germany]
+ L-glutamine [GIBCO Invitrogen, Karlsruhe, Germany]
methionine sulphoximine [Sigma-Aldrich, Taufkirchen, Germany]
0.5 % Trypsin, 5.3 mM EDTA, [GIBCO Invitrogen, Karlsruhe, Germany]
20 mM Tris, pH 7.4, 5 mM MgCl2, 1 % Triton X 100, 150 mM NaCl
2.1.5.3 Antibiotics
Ampicillin, 1000 x
Penicillin/Streptomycin, 100 x
Zeocin™
Kanamycin, 1000 x
Puromycin
2.1.6
100 mg/ml stock solution in H2O [Roth, Karlsruhe, Germany]
104 U/ml / 104 µg/ml [GIBCO Invitrogen, Karlsruhe, Germany]
[Invitrogen, Karlsruhe, Germany]
Solutions
Blotting buffer
Bradford Solution, 5 x
Carbonate-bicarbonate
buffer
Coomassie
Crystal violet
DNA loading buffer, 5 x
ECL reagent: solution A
ECL reagent: solution B
Glutaraldehyde
Glycogen
20 % methanol, 192 mM glycine, 25 mM Tris, pH 8.3
Bio-Rad protein assay [Bio-Rad, Munich, Germany]
50 mM, pH 9.6, (3 ml of 0.1 M sodium carbonate, 7 ml of 0.1 M
sodium bicarbonate)
Serva Blau G C.I. 42655, Brilliant Blue G-250 [Serva Feinbiochemica,
Heidelberg, Germany], 80 mg in 1 L H2O, 35 mM HCl
0.5 % crystal violet, 20 % methanol in H2O
1 ml TAE buffer, 50 x, 2.5 ml glycerol, 0.02 % (w/v) bromophenol blue,
ad 10 ml H2O
0.1 M Tris/HCl (pH 8.6), 1.25 mM luminol sodium salt [Sigma-Aldrich,
Taufkirchen, Germany]
11 mg para-hydroxycoumaric acid [Sigma-Aldrich, Taufkirchen,
Germany] in 10 ml DMSO
25 % solution in H2O [Serva, Feinbiochemica, Heidelberg, Germany]
#R0561 [Fermentas, St. Leon-Rot, Germany]
32
Kirstin A. Zettlitz
IMAC Na-phosphate buffer,
5 x (low salt)
Lysis-buffer
PBA
PBS, 1 x
PEG-NaCl
Periplasmatic preparation
buffer, PPB
Protein A buffer A
Protein A elution buffer
Protein A wash buffer
SDS loading buffer, 5 x
SDS running buffer, 10 x
Sulfo-NHS-SS-Biotin
TAE buffer, 50 x
TMB substrate
TNF
2.1.7
E. coli HB2151
E. coli DH5α
E. coli BL21-DE3
Phage VCS M13
B16-FAP
Colo+
MEF
HEK 293
HT1080
HeLa
Antigen
0.02 M NaH2PO4 (2.4 g), 0.15 M NaCl (8.8 g) ad 1 L H2O, pH 8.0
100 mM glycine pH 3.0
100 mM Tris-HCl pH 8.0 and 10 mM Tris-HCl pH 8.0
non-reducing: 30 % (v/v) glycerol, 3 % (w/v) SDS, 2 µg/ml
bromophenol blue in 62.5 mM Tris-HCl, pH 6.8
reducing: add 5 % (v/v) -mercaptoethanol
1.92 M glycine, 0.25 M Tris, 1 % SDS, pH 8.3
Prod.no. 21328 [Pierce, Rockford, USA]
2 M Tris, 0.95 M glacial acetic acid, 50 mM EDTA in H2O, pH 8
100 µl TMB (100 mg/ml stock in DMSO), 2 µl 30 % H2O2, 10 ml Naacetate buffer, pH 6 (100 mM)
soluble human TNF + HSA; 0.5 mg/ml (21.07.2008), in-house
production
[Stratagene, La Jolla, USA], Genotype: supE thi-1 Δ(lac-proAB) Δ(mcrB-hsdSM)5
(rK– mK–) [F´ traD36 proAB lacIqZΔM15].
(Carter et al. 1985)
F- endA1 glnV44 thi-1 recA1 relA1 gyrA96 deoR nupG Φ80dlacZΔM15
Δ(lacZYA-argF)U169, hsdR17(rK- mK+), λ–
F– ompT gal dcm lon hsdSB(rB- mB-) λ(DE3 [lacI lacUV5-T7 gene 1 ind1
sam7 nin5])
Interference-resistant helper phage, cat.no. 200251 [Stratagene, La Jolla, USA]
Cell lines
CHO-K1
Kym-1
2.1.9
250 mM Na-phosphate (37.38 g Na2HPO4 · 2H2O + 6.24 g NaH2PO4 ·
2H2O), 1.25 M NaCl, pH 7.5, ad 1 L H2O
100 mM NaH2PO4, 10 mM Tris, 8 M urea
2 % FCS, 0.2% NaN3 in sterile PBS, 1 x
2.67 mM KCl, 1.47 mM KH2PO4, 137.93 mM NaCl, 8.06 mM
Na2HPO4 · 7H2O, pH 7.5
200 g Polyethylenglycol 6000, 2.5 M NaCl, ad 1 L H2O
30 mM Tris-HCl pH 8.0, 1 mM EDTA, 20 % sucrose in H2O
Bacteria and phage
E. coli TG1
2.1.8
Methods
Chinese hamster ovary [Lonza Biologics, Berkshire, UK]
Human rhabdomyosarcoma from neck tumor (JCRB0627), supplied by M Sekignchi,
University of Tokyo, Japan
Mouse melanoma
Human adenopancreatic carcinoma (Colo 357), provided by [Multimmune GmbH,
Munich, Germany]
immortalized mouse embryonic fribroblasts from TNFR1/TNFR2 double knock-out mice,
stably transfected with human TNFR1-Fas or TNFR2-Fas (Krippner-Heidenreich et al.
2002)
Human embryonic kidney
Human fibrosarcoma (ECACC85111505)
Human cervix carcinoma (DSMZ ACC 57)
Antibodies
Origin
human
Conjugate
-
Company
[Sigma-Aldrich, Taufkirchen, Germany]
33
Cat.no.
I 4506
Kirstin A. Zettlitz
His6-Tag, Hisprobe (H-3)
Hsp70
(cmHsp70.1)
Human Albumin
Human IgG (Fabspecific)
Human IgG (Fcspecific)
Human IgG
(whole molecule)
Human lambda
light chain-b+f
Human TNFReceptor I
(CD120a)
M13
Mouse IgG (Fcspecific)
Myc-tag (9E10)
Methods
IgG
mouse
HRP
mouse
FITC
rabbit
goat
HRP
[Santa Cruz Biotechnology, Santa Cruz, sc-8036
USA]
[Multimmune
GmbH,
Munich,
Germany]
A 0433
A 0293
goat
HRP
A 0170
rabbit
HRP
A 8792
goat
HRP
[Bethyl, Montgomery, USA]
A80-116P
mouse
-
[Hbt Hycult biotechnology b.v.]
in house production
Cat.no.
HM2020b
goat
goat
HRP
HRP
[Pharmacia, Stockholm, Sweden]
27-9421-01
A 2554
mouse
-
HRP
OM-11-908
(Genosys)
A 0545
Rabbit IgG (whole goat
molecule)
2.1.10 Antigens
CEA
HSA
Hsp70
FZ30-AC25 [Europa Bioproducts Ltd, Cambridge, UK]
A3782 [Sigma-Aldrich, Taufkirchen, Germany]
[Multimmune GmbH, Munich, Germany]
2.1.11 Enzymes
Alkaline phosphatase
FailSafe™PCR
Herculase®Enhanced
DNA Polymerase
REDTaq ReadyMix™
Taq DNA-Polymerase
T4 DNA Ligase
N-glycosidase F
5 U/µl [Fermentas, Burlington, USA]
PreMixSelection Kit, Cat.Nos. FS99060 [Epicentre, Madison, USA]
5 U/µl [Stratagene, La Jolla, USA]
0.06 U/µl, Cat.no. R2523 [Sigma-Aldrich, Taufkirchen, Germany]
1 U/µl [Fermentas, Burlington, USA]
5 U/µl), [Fermentas, Burlington, USA]
PNGase F, cat.no. 11 365 185 001 [Roche Diagnostics, Mannheim, Germany]
2.1.12 Restriction Enzymes
AdeI/DraIII
Alw44I/ApaLI
BamHI
Bpu10I
BshTI/AgeI
Bsp1407I/BsrGI
Cfr9I/XmaI
EcoRI
HindIII
10 U/µl
10 U/µl
10 U/µl
10 U/µl
10 U/µl
10 U/µl
10 U/µl
10 U/µl
10 U/µl
[Fermentas, Burlington, USA]
34
Kirstin A. Zettlitz
KpnI
NcoI
NheI
NotI
PstI
SfiI
SgsI (AscI)
XbaI
XhoI
20 U/µl
10 U/µl
10 U/µl
10 U/µl
20 U/µl
10 U/µl
5 U/µl
10 U/µl
10 U/µl
Methods
[New England Biolabs, Ipswich, USA]
[New England Biolabs, Ipswich, USA]
2.1.13 Kits, Marker
Amine Coupling Kit
First Strand cDNA Synthesis Kit
Gene RulerTM DNA Ladder Mix ready-touse
High sensitivity human ELISA set
(interleukin 6, interleukin 8)
MycoAlert®Mycoplasma Detection Kit
NucleoBond®Xtra Midi
NucleoSpin®Extract II Kit
Page RulerTM Prestained Protein Ladder
Page RulerTM Prestained Protein Ladder
Plus
PureLink™ Quick Plasmid Miniprep Kit
QIAprep® Spin Miniprep Kit
Streptavidin Poly-HRP
Prod.no. 3501-3001 [Attana, Stockholm, Sweden]
#K1612 [Fermentas, St. Leon-Rot, Germany]
#SM0333 [Fermentas, St. Leon-Rot, Germany]
Cat.No.: 31330069/ 31330089 [ImmunoTools, Friesoythe,
Germany]
Cat.no.LT07-218 [Lonza Rockland, Rockland, USA]
[Macherey-Nagel, Düren, Germany]
[Macherey-Nagel, Düren, Germany]
Catalog nos. K2100-10, K2100-11 [Invitrogen, Carlsbad,
USA]
Cat no 27104 [Qiagen, Hilden, Germany]
Cat.no. 31334248 [ImmunoTools, Friesoythe, Germany]
2.1.14 Primer
2.1.14.1 Primer for cloning of IgG humex
name
HindIII-Igk-AgeIVL-humex-back
no sequence
target
242 5’AAT CCC AAG CTT ATG GAG ACA GAC ACA CTC CTG CTA VL
TGG GTA CTG CTG CTC TGG GTT CCA GGT TCC ACC GGT humex
VL humex-AscIforward
HindIII-Igk-AgeIVH-humex-back
240 5’ T TGG CGC GCC CTT TGG CTG GCC CAG CAC GGT CAG VL
CTT GGT C 3’
humex
5’AAT
CCC
AAG
CTT
ATG
GAG
ACA
GAC
ACA
CTC
CTG
CTA
243
VH
TGG GTA CTG CTG CTC TGG GTT CCA GGT TCC ACC GGT humex
CAG GCC GTG GTG ACC CAG GAG 3’
GAG GTG CAG CTG GTC GAG AGC 3’
VH humex-XhoI- 241 5’ T ACC GCT CGA GAC GGT CAC GGT GGT GCC CTG 3’
for
VH
humex
2.1.14.2 Primer for cloning of Hsp70 fragments
name
Hsp70-495-NotI-for
no
235
Hsp70-504-NotI-for
270
Hsp70-514-NotI-for
234
Hsp70-Fragment 1-
198
sequence
5’ A TAG TTT AGC
CGT GGC 3’
5’ A TAG TTT AGC
CTT GTT GGC 3’
5’ A TAG TTT AGC
CAG GCG 3’
5’ A TAG TTT AGC
GGC CGC GGT GCT CTT GTC
target
Hsp70
GGC CGC GGT GAT GGT GAT
Hsp70
GGC CGC CTC CTC CTT GCT
Hsp70
GGC CGC GTC CAG CAG CAG
Hsp70
35
Kirstin A. Zettlitz
NotI-for
Hsp70-Fragment 3-NotIfor
Hsp70-Fragment 4-NotIfor
Hsp70-Fragment2-NotIfor
NcoI-Hsp70-425-back
Methods
194
CAG GTC 3’
5’ A TAG TTT AGC GGC CGC GTT GAA GGC GTA
GGA CTC 3’
Hsp70
192
5’ A TAG TTT AGC GGC CGC ATC TAC CTC CTC
AAT GGT G 3’
Hsp70
196
5’ A TAG TTT AGC GGC CGC CTC GAT CTG GGG
CAC GCC 3’
Hsp70
233
5’ CAT GCC ATG GCC ACG CAG ATC TTC ACC ACC
3’
5’ CAT GCC ATG GCC ACG GCC GGA GGC GTG ATG
3’
5’
CAT GCC ATG GCC ACC TAC TCC GAC AAC
CAA 3’
5’ CAT GCC ATG GCC ATG ACG AAA GAC AAC AAT
3’
5’ CAT GCC ATG GCC GGC ATC CCT CCG GCC CCC
AGG 3’
5‘ CAT GCC ATG GCC CAG ATC GAG GTG ACC TTC
3’
5’ CAT GCC ATG GCC AAC GGC ATC CTG AAC 3’
5’ CAT GCC ATG GCC ACG GAC AAG AGC ACC 3’
5’ CAT GCC ATG GCC AAA GCC GCG GCG ATC GGC
3’
Hsp70
NcoI-Hsp70-404-back
232
NcoI-Hsp70-430-back
260
NcoI-Hsp70-449-back
266
NcoI-Hsp70-463-back
267
NcoI-Hsp70-473-back
268
NcoI-Hsp70-482-back
NcoI-Hsp70-490-back
NcoI-Hsp70-Fragment 1back
269
261
199.1
Hsp70
Hsp70
Hsp70
Hsp70
Hsp70
Hsp70
Hsp70
Hsp70
197.1 5’ CAT GCC ATG GAC AAG TCC GAG AAC GTG 3’
Hsp70
195.1 5’ CAT GCC ATG GTG CTG ATC CAG GTG TAC 3’
Hsp70
193.1 5’ CAT GCC ATG GTG TCA GCC AAG AAC GCC 3’
Hsp70
2.1.14.3 Primer for site-directed-mutagenesis of scFvIZI-06.1
name
CDRH1mutbackNheI
CDRH2mutbackXmaI
CDRL2-mutBamHI-for
CDRL1-mutPstI-for
no
295
sequence
5‘ TGC AAA GCT AGC GGC TAT ACC TTT ACC NHK NHK NHK
ATT AAC TGG GTG CGT CAG GCA CCC 3‘
target
VH IZI06.1
296
5’ CAG GCA CCC GGG CAG GGC CTG GAA TGG ATT GGC NHK
ATT NHK CCG NHK NHK GGC NHK GCA NHK TAC AAC NHK
AAA TTC AAA GCG CGT GTG ACC 3’
VH IZI06.1
297c 5’ GCT ACC GGA TTC GCT AAA GCG ATC CGG CAC GCC MDN
VL IZI06.1
VL IZI06.1
AAA ACG MDN GCT CAC MDN ATA AAT CAG CAG CTG CGG 3’
298
5’ CGG TTT CTG CAG ATA CCA ATG CAG MDN GGT MDN GCC
MDN MDN MDN CAG CAG GCT CTG GCT GCT ACG GCA 3’
2.1.14.4 Primer for screening or sequencing
name
fdSeq1
LMB2
LMB3, M13-RP
Lonza-14.4-R
Lonza-F
pET-Seq1, T7-981079
pSec-Seq2
no
258
326
88
84
82
89
91
sequence
5’
5’
5’
5’
5’
5’
5’
GAA
GTA
CAG
CCT
GCC
TAA
TAG
TTT
AAA
GAA
GAA
ACC
TAC
AAG
TCT
CGA
ACA
CCT
AGA
GAC
GCA
GTA
CGG
GCT
GAA
CAT
TCA
CAG
TGA
CCA
ATG
ACA
AAT
CTA
TCG
GG 3’
GT 3’
ACC A 3’
TAA 3’
AGC 3’
TAG G 3’
AGG 3’
target
pHEN
pAB1
pAB1, pHEN
pEE6.4, pEE14.4
pEE6.4, pEE14.4
pSecTagA
pSecTagA
36
Kirstin A. Zettlitz
Methods
2.1.15 Vectors
pAB1
pEE14.4
pEE6.4
pET-28b
pHEN2
pSecTagA
3342 bp, vector for prokaryotic protein expression in E. coli TG1 (Kontermann
et al. 1997)
10124 bp, GS-encoding expression vector, GS-System™pEE Expression Vectors
[Lonza Biologics, Berkshire, UK]
5067 bp, accessory vector, GS-System™pEE Expression Vectors [Lonza
Biologics, Berkshire, UK]
5369 bp, Cat-no. 69685-3 [Novagen, Merck Bioscience, Nottingham, UK]
4600 bp, 15 aa linker, pUC119 backbone
5166 bp, eukaryotic expression [Invitrogen, Karlsruhe, Germany]
2.2 Methods
2.2.1
Cloning
2.2.1.1 Bacterial culture
E. coli strains TG1 and DH5α were used as cloning hosts and for amplification of plasmid DNA. E. coli
strains TG1, BL21-DE3 and HB2151 were used for production. Glycerol stocks consisting of 700 µl
overnight culture and 300 µl glycerol were frozen at -80 °C for long-time storage.
2.2.1.2 Chemical competent E. coli cells
50 ml LB medium were inoculated with 1 ml of an overnight culture of E. coli TG1, BL21-DE3 or DH5α
and incubated at 37 °C to OD600 nm 0.5 – 0.6. The cells were cooled on ice for 15 min and harvested by
centrifugation (Jouan GR4i, 3500 rpm, 5 min, 4 °C). The resulting cell pellet was resuspended in 50 ml
0.1 M CaCl2 and incubated on ice for 30 minutes. The cells were centrifuged again (3500 rpm, 5 min,
4 °C) and resuspended in 10 ml 20 % glycerol/50 mM CaCl2. Finally the cell suspension was frozen in
aliquots of 500 µl and stored at -80 °C.
2.2.1.3 Electro competent E. coli cells
450 ml SOB medium were inoculated with 5 ml of an overnight culture (E. coli TG1, SOB) and
incubated at 37 °C for about 3 h with vigorous shaking until the OD600 nm was approximately 0.7 – 0.8.
Cells were chilled on ice for 1 h and harvested by centrifugation (J2-MC, JA10, 2000 x g, 10 min, 4 °C).
The cell pellet was resuspended in 200 ml cold sterile water and again centrifuged (J2-M2, JA10, 2000
x g, 15 min, 4 °C). The pellet was resupended in 200 ml cold sterile water and incubated on ice for 30
min. The cells were harvested by centrifugation (J2-MC, JA10, 2000 x g, 15 min, 4 °C) and this time
resuspended in 25 ml cold 10 % glycerol and transferred to a 50 ml conical tube. The cell suspension
was incubated for additional 30 min on ice and cells were harvested by centrifugation (Jouan GR4i,
1500 x g, 15 min, 4 °C). The supernatant was removed and cells were resuspended in a final volume
of 1 ml cold 10 % glycerol. 40 µl aliquots of electro competent cells were directly used for
electroporation.
37
Kirstin A. Zettlitz
Methods
2.2.1.4 Polymerase chain reaction
Polymerase chain reaction (PCR) was used for amplification of desired DNA fragments. Different
Polymerases and different composition were used:
Polymerase
Input template
Primer (each)
Deoxynucleotide
buffer
MgCl2
dH2O
total
volume
0.5 µl Herculase Enhanced
DNA Polymerase (2.5 U
total)
1-15 ng vector DNA
100-200 ng genomic DNA
1.25 µl Taq DNA
Polymerase (1.25 U
total)
1-15 ng vector DNA
100-200 ng genomic
DNA
1 µl (10 pmol/µl)
1 µl (10 pmol/µl)
(1 µM final concentration) (1
µM
final
concentration)
200 µM each dNTP (0.8 200 µM each dNTP
mM total)
(0.8 mM total)
0.5 µl FailSafe PCR
Enzyme Mix (1.25 U total)
1-15 ng vector DNA
100-200 ng genomic DNA
0.5 µl (50 pmol/µl)
(1 µM final concentration)
PreMix contains 400 µM
of each dNTP (0.8 mM
total)
5 µl 10 x Herculase 5 µl 10 x Taq buffer PreMix contains 100 mM
Reaction buffer
(+(NH4)2SO4)
Tris-HCl, pH 8.3, 100 mM
KCl
4 µl (25 mM)
(2
mM
final
concentration)
x µl
x µl
x µl
reaction 50 µl
50 µl
25 µl
2.2.1.5 Site directed mutagenesis
In PCR the randomized primers amplified most of the scFv gene and introduced mutations into the
CDR. At the same time these primers appended the restriction sites for cloning the fragment into
acceptor plasmid pHENIS-scFvIZI-06.1_notwt (midi-no 0586) which was modified to contain a
deletion after CDRH2 and an additional stop-codon to prevent production of wild type scFv. 50 µl
PCR reaction mix was prepared containing:
water
34.25 µl
dNTPs (20 mM, 5 mM each)
2.5 µl
10 x taq buffer +(NH4)2SO4
5 µl
MgCl2 (25 mM)
4 µl
primer forward (10 pmol/µl)
1 µl
primer reverse (10 pmol/µl)
1 µl
scFv gene template (100 ng)
1 µl
taq-DNA polymerase (1 U/µl) 1.25 µl
38
Kirstin A. Zettlitz
Methods
PCR program no 14 was used (94°, 1’, 55°, 1’, 72°, 1’; 30 cycles). The gene-repertoires were gelpurified (1 % agarose gel electrophoresis) and the DNA was extracted using NucleoSpin®Extract II Kit
(Macherey-Nagel). Purified DNA was dissolved in 30 µl H2O and DNA concentration was determined
by photometric measurement (Nanodrop).
2.2.1.6 Restriction digest, Ligation, Ethanol precipitation
1 µg DNA in a total volume of 20 µl was used in analytical digestion, while 5 – 10 µg DNA in a total
volume of 50 µl was used in preparative digestion. Restriction enzymes were used in excess (ca. 20
U/ reaction mix). The used buffers and incubation times were chosen according to the
manufacturer’s protocol. For buffer exchange the NucleoSpin® Extract II Kit (Machery-Nagel) was
used. Previous to ligation, vectors were dephosphorylated with 1 µl of alkalic phosphatase (5 U/µl) in
a total volume of 50 µl alkalic phophatase buffer for 60 min at 37°C to avoid religation of the vector.
Ligation of linearised, dephosphorylated vector and insert was carried out in a total volume of 20 µl
ligation buffer with a molar ration of vector to insert of 1:3 to 1:5 and 1 µl T4-DNA ligase (5 U/µl).
After incubation for 60 min at room temperature 10 µl of the ligation mixture were used for
transformation of competent bacterial cells.
To digest scFv repertoires 50 µl reaction mixes were prepared containing 1 – 4 µg scFv DNA, one or
two restriction enzymes (1 – 2 µl each) and 5 µl of the appropriate buffer 10 x (according to
manufacturer’s protocol). The digestion was incubated overnight at 37 °C and gel-purified the next
day. Approximately 20 µg of purified pHEN2 scFvIZI-06.1_notwt were digested with the appropriate
enzymes and dephosphorylated using CIP (calf intestine phosphatise, Fermentas). For ligation of scFv
repertoires 2000 ng digested acceptor vector and 5-fold molar excess of digested scFv gene
repertoire (molar ratio of insert to vector: 5:1) were mixed, 3 µl T4 DNA ligase (5 U/µl) and 5 µl of 10
x ligation buffer were added (total volume: 50 µl). Ligation was carried out overnight at 16 °C.
DNA was ethanol precipitated by adding 1/10 volume (5 µl) of 3 M NaOAc pH 5.2 and glycogen
(Fermentas) to a final 0.05 – 1 µg/µl concentration. 2.5 volume (125 µl) of 100 % EtOH were added,
mixed and the mix was incubated for 1 hour at -80 °C. After centrifugation (eppendorf, 13000 x g,
4 °C) the DNA pellet was rinsed two times with 70 % EtOH, air-dried and resuspended in 10 µl H2O.
2 µl/transformation of were used for electroporation.
2.2.1.7 Antibody humanization
A humanized version of cmHsp70.1 (humex) was generated by grafting all 6 complementarity
determining regions (CDRs) onto human variable germline genes IGHVH3-66 (DP86) and IGLV7-43
(DPL18, VL3-2) similar to the tumex VH and VL sequence, respectively. The amino acid sequences
39
Kirstin A. Zettlitz
Methods
were back-translated into a nucleotide sequence using preferred codon frequencies of homo sapiens.
Codon-optimized DNA encoding the two humanized variable domains (VH humex, VL humex) was
synthesized by GeneArt (Regensburg, Germany). The genes encoding the variable heavy and light
chain domain of antibody humex were supplied in vector pGA4 containing appropriate cloning sites.
For cloning of scFv humex, plasmid pGA4-VHhumex was digested with restriction enzymes NcoI and
XhoI and the resulting fragment was cloned into vector pHENIS (pHEN2 with His-tag, containing a
linker sequence (G4S)3) digested with the same enzymes. Subsequently, the plasmid pGA4-VLhumex
was digested with the restriction enzymes ApaLI and NotI and the resulting fragment was cloned into
vector pHENIS-VHhumex digested with the same enzymes. Finally, the DNA encoding the scFv
fragment was subcloned into pAB1 via NcoI and NotI.
2.2.1.8 Cloning of IgG humex
For construction of an IgG molecule in the Lonza expression system, the VH humex-encoding region
was reamplified from plasmid pGA4 with primers incorporating restriction sites HindIII and XhoI and
a modified Igκ-leader sequence containing an AgeI site to allow correct N-terminal processing
(HindIII-Igκ-AgeI-VHhumex-back, VHhumex-XhoI-for) and cloned into vector pEE6.4-CH1-CH3
containing the human γ1 heavy chain constant regions. The VLhumex encoding region was
reamplified from plasmid pGA4 with primers incorporating both restricition sites HindIII and AscI and
the Igκ-leader sequence (HindIII-Igκ-AgeI-VLhumex-back, VLhumex-AscI-for) and cloned into pEE14.4Cλ containing the human lamda light chain constant region. The two plasmids were then combined
according to the manufacturer’s protocol (via NotI and BamHI) resulting in a bicistronic expression
plasmid with the heavy chain gene downstream of the light chain gene (pEE14.4 IgG humex).
2.2.1.9 Cloning of Hsp70 fragments
Human Hsp70 cDNA was reverse-transcribed from the total RNA of heat-shock treated Colo+ cells
and used as PCR-template for amplification of overlapping Hsp70 fragments. The respective primers
introduced a NcoI and a NotI restriction site at the 5’ and the 3’ ends. The PCR products were
digested with restriction enzymes NcoI and NotI and cloned into plasmid pET-28b (Novagen)
containing a C-terminal hexahistidyl-tag sequence. The expression plasmids were transformed into
chemically competent E. coli BL21-DE3. Hsp70 fragments less than 100 amino acids were produced as
fusion proteins with human serum albumin domain 3 (AlF3: aa 382-582 of HSA). The DNA encoding
AlF3 was cloned into vector pAB1 as NotI-AlF3-His-EcoRI fragment resulting in pAB1-AlF3. The Hsp70
fragments were introduced upstream of AlF3 via NcoI/NotI, the resulting plasmids were transformed
into E. coli TG1.
40
Kirstin A. Zettlitz
Methods
2.2.1.10 Cloning of chimeric TNFR1-Fc fusion proteins
DNA encoding the extracellular region of human TNFR1 (aa 29-211), rhesus TNFR1 (aa 27-209), and
mouse TNFR1 (aa 30-212) was produced synthetically (Geneart, Regensburg, Germany), introducing
appropriate restriction sites between the individual domains, and cloned into pSecTagL1-Fc
(modified from pSecTag-FcHis, (Muller et al. 2008). Chimeric human/mouse TNFR1-Fc fusion proteins
were generated by exchanging the different regions between human and mouse TNFR1-Fc.
2.2.1.11 Heat shock transformation of E. coli TG1
Chemical competent cells were thawed on ice and 100 µl of cell suspension were incubated together
with 10 µl ligation for 15 min on ice. The bacteria were exposed to a heat shock for 45 sec in a 42 °C
water bath and then cooled for 1 min on ice. After addition of 1 ml LB medium the bacteria were
grown for 1 hour at 37 °C to permit expression of the resistance gene. Cells were harvested by
centrifugation, resuspended in the reflux (ca. 100 µl) and plated on agar plates (LB
amp, glc).
Plates
were incubated overnight (37 °C).
2.2.1.12 Electroporation of E.coli TG1
Electroporation cuvettes, microcentrifuge tubes were pre-chilled and electro competent cells were
handled on ice. 40 µl E. coli TG1 were mixed with 2 µl plasmid DNA (in H2O), the electroporator was
set to a voltage of 1700 V (17 kV/cm field strength), a resistance of 200 Ω and a capacity of 25 µF.
The sample was pulsed once and immediately resuspended in pre-warmed SOC medium (1 ml, 37
°C). Cells were transferred to a sterile 14-ml BD Falcon polypropylene round-bottom tube and
incubated for 1 hour at 37 °C with shaking. Serial dilutions of the transformation mix were plated
onto TYEamp, glc plates. The cells were harvested by centrifugation, resuspended in 200 µl SOC medium
and plated onto 15 cm TYEamp,
glc
plates. Plates were incubated overnight at 37 °C. The next day
colony numbers were counted for calculation of the library size. Bacteria were scraped from the
plates by adding 2 ml of 2xTY medium. The number of bacteria per ml was calculated (OD600nm of 1.0
corresponds to 5 x 108 cells). Glycerol stocks containing bacteria in 10-fold excess of the calculated
number of different clones were stored at – 80 °C.
2.2.1.13 Screening of clones
The screening for positive clones was performed by analytical PCR for inserts. Single clones were
picked and used as template in REDTaq™Ready Mix (Sigma), simultaneously this clones were plated
on a masterplate and incubated overnight (37 °C). Both PCR program and primer were chosen
according to the expected fragments and the used plasmids. Samples were separated in agarose gel
electrophoresis and positive clones could be identified due to bands of the predicted size. One of
these clones was used to inoculate an overnight culture designated for plasmid isolation (Midi).
41
Kirstin A. Zettlitz
Methods
2.2.1.14 Plasmid-DNA Isolation (Midi, Mini)
For the isolation of plasmid DNA from bacterial cells the NucleoBond®Xtra Midi (Macherey-Nagel)
was used according to the manufacturer’s instruction. The obtained DNA pellets were air dried
resolved in 100 µl H2O and stored at -20 °C.
Purification of plasmid DNA from 1 – 5 ml overnight culture was done using QIAprep® Spin Miniprep
Kit (Qiagen) according to manufacturer’s instructions.
2.2.1.15 Sequence Analysis
Sequencing was performed by GATC Biotech AG (Konstanz, Germany). Analysis of the resulting
sequence was performed using the program `blast` (Tatusova and Madden 1999) and the program
‘Clone Manager 7’ version 7.11 for windows.
2.2.2
Phage display
2.2.2.1 Preparation of helper phage
An overnight culture was inoculated with a single clone of TG1 grown on a minimal plate. The next
day this overnight culture was used to inoculate 12 ml 2xTY. The culture was incubated at 37 °C with
shaking until OD600nm reached 0.3 – 0.5. 100 µl of serial dilutions of helper phage (VCSM13,
Stratagene) were mixed with 100 µl of log-phase TG1 and 3 ml pre-warmed (42°C) top agar and
plated onto TYE plates. The next day a single plaque from the lawn was transferred to 3 ml 2xTY
containing 100 µl of a TG1 overnight culture and incubated for 3 hours with shaking at 37 °C. The
grown plaque was used to inoculate 500 ml 2xTY and incubated for 1 hour (37 °C, shaking) before 25
µg/ml kanamycin was added. The culture was incubated overnight at 37 °C with shaking. The next
day bacteria were removed by centrifugation (J2-MC, JA10, 6300 x g, 30 min) and the supernatant
containing the helper phage was heated to 65 °C for 15 min. After spinning down cell debris, helper
phage were aliquoted and stored at -20 °C. An aliquot was checked on a TYE plate for remaining
bacteria. Phage titre was determined using top agar and serial dilution of phage.
2.2.2.2 Immunotube selection
Immunotubes were coated with 1 ml of antigen (1 – 0.1 µg/ml) in PBS overnight at 4 °C. The next day
immunotubes were washed (PBS, 3 x) and blocked with PBS, 2 % skimmed milk powder (2 % MPBS)
for 2 hours at RT. Antibody-phage (from the library-stock or from a previous selection round) were
pre-incubated with 1 ml 2 % MPBS for 30 min. Pre-blocked phage were incubated in the immunotube
for 2 hours. Immunotubes were washed with PBS, 0.1 % Tween 20 (PBST) and PBS with increasing
stringency (Table 2-1) and phage were eluted by incubation with 1 ml 100 mM TEA (triethylamine)
for 8 – 10 min. Eluted phage were transferred to a sterile reaction tube containing 500 µl 1 M Tris-
42
Kirstin A. Zettlitz
Methods
HCl pH 7.5 to neutralize the solution. 1 ml of neutralized phage was added to 10 ml of log-phase TG1
and (from round 2 on) 10 µl of neutralized phage were added to 1 ml of log-phase HB2151 (to get
colonies for soluble expression). Transduced bacteria were incubated at 37 °C standing for 30 min
and additional 30 min with shaking. Serial dilutions of TG1 and HB2151 (in 100 µl 2xTY) were plated
onto TYEamp, glc plates. The remaining TG1 culture was centrifuged (Jouan GR4i, 2100 x g, 10 min) and
the cell pellet was resuspended in 1 ml 2xTY and plated onto nunc square 24x24 cm TYEamp, glc plates.
Table 2-1: Conditions for immunotube selection using immobilized antigen.
wash PBST
wash PBS
10 µl = 2 x 10
20 x
20 x
2 h, RT
1 µl = 5 x 109
1.5 h, 6 x
20 x
2 h, RT
1 µl = 5 x 109
1.5 h, 6 x
20 x
Selection
Round
antigen (nM)
incubation
phage
1st
10 nM (1 µg/ml)
2 h, RT
2nd
1 nM (0.1 µg/ml)
3rd
1 nM (0.1 µg/ml)
11
2.2.2.3 Equilibrium selection
Phage were pre-blocked (2 % MPBS, 30 min, RT), for the first round of selection the number of phage
should be at least 100 times higher than the library size. Biotinylated antigen was added directly into
equilibrated phage to a final concentration of 10 nM (following rounds of selection see Table 2-2).
The mix was incubated for 1 hour at RT on a rotator. 500 µg of washed and blocked streptavidinmagnetic beads (Dynabeads) were added to the phage-antigen mix and incubated for 15 min on a
rotator. A magnetic separator was used to separate the beads. The beads were washed 3 times in
MPBST (2 % MPBS, 0.1% Tween 20) and subsequently in PBS (following rounds of selection see Table
2-2) and transferred to a new reaction tube. The phage-antigen complex was eluted with 200 µl 10
mM DTT (5 min, rotator). 1 ml PBS was added, the beads were separated and the supernatant was
transferred to a new tube. 750 µl of eluted phage were used to inoculate 10 ml log-phase E. coli TG1
and 10 µl eluted phage were used to inoculate 1 ml of log-phase E. coli HB2151 in an analogous
manner as described in 2.2.2.2.
Table 2-2: Conditions for equilibrium selection using biotinylated antigen.
Selection
Round
0
1st
2nd
3rd
4th
5th
6th
antigen (nM)
incubation
phage
moTNFR1-Fcbiotin, 20 nM
10 nM
10 nM
1 nM
1 nM
0.1 nM
0.1 nM
1 h, RT
10 µl = 2x10
4 h, RT
o/n, 4°C; 1 h, RT
o/n, 4°C;1h, RT
3 h, RT
o/n, 4°C; 1h, RT
3 d, 4°C; 1h, RT
10 µl = 2x10
10
10 µl = 4x10
9
1 µl = 4x10
9
1 µl = 4x10
9
1 µl = 4x10
9
1 µl = 4x10
11
11
Dyna
beads
500 µg
500 µg
500 µg
100 µg
100 µg
100 µg
100 µg
wash MPBST (PBS)
=> use supernatant
selection
3 (1) x
15 (5) x
20 (10) x
10, 1h, RT (10) x
10, 2h, RT (10) x
10, 2h, RT (10) x
43
for
Kirstin A. Zettlitz
Methods
2.2.2.4 Rescue of phage libraries for selection with helper phage
Transduced TG1 were scraped from the nunc square plates by adding 10 ml 2xTY medium and using a
glass spreader. Library stocks (700 µl bacteria and 300 µl of 50 % glycerol, 0.2 µm sterile filtered)
were stored at – 20 °C. The number of bacteria per ml was calculated (OD600 nm of 1.0 corresponds to
approx. 5 x 108 cells). The input of cells for rescuing was calculated to be in 100-fold excess of
different clones in the library but should not exceed a starting OD600
nm
of about 0.1. A typical
inoculum was 20-50 µl of bacteria in 50 ml 2xTY (100 µg/ml ampicillin, 2 % glucose). The culture was
incubated at 37 °C with shaking until an OD600
nm
of 0.4 – 0.5 is reached (1-2 x 1010 bacteria).
Approximately 10-fold excess (~1011 t.u.) of helper phage was added and incubated for 30 min at
37 °C standing and additional 30 min at 37 °C with shaking. 1 µl was plated onto TYE (100 µg/ml
kanamycin) plate to check for infectivity of helper phage. Transduced cells were harvested by
centrifugation (Jouan GR4i, 3000 x g, 10 min) to remove glucose, resuspended in 50 ml 2xTY (100
µg/ml ampicillin, 25 µg/ml kanamycin, no glucose) and incubated overnight at 30 °C with shaking.
The next day bacteria were removed by centrifugation and the supernatant containing the amplified
antibody-phage was transferred into a 500 ml centrifuge bottle. Phage were precipitated by adding
1/5 volume of 20 % PEG, 2.5 M NaCl and incubation at 4 °C for 1 hour with stirring, subsequently
phage were harvested by centrifugation and resuspended in 40 ml PBS. Remaining bacteria were
removed by an additional centrifugation step. PEG precipitation was repeated and after
centrifugation the white pellet was resupended in 1 ml PBS. Phage titre was determined as described
in 2.2.2.1. The phage stock was used at once for the next round of selection or stored at 4 °C for
approximately 1 week.
2.2.2.5 Polyclonal phage ELISA
ELISA plates were coated with antigen (hu/mo TNFR1-Fc) at 0.5 µg/ml in PBS overnight at 4 °C. After
blocking (2 h, 2 % MPBS, RT) 10 µl of rescued phage in 100 µl 2 % MPBS were added and incubated
for 1 hour at room temperature (RT). Bound phage were detected by anti-M13 antibody-HRP diluted
1/5000 in 2 % MPBS (1 h, RT) as described in 2.2.9.
2.2.2.6 Screening of soluble scFv by ELISA
Single HB2151 colonies from selection from phagemid libraries are transferred to a sterile 96-well
microtiter plate containing 100 µl 2xTY (100 µg/ml amp, 2 % glucose). This masterplate was
incubated overnight at 37 °C with shaking and used the next day to inoculate a fresh plate containing
125 µl 2xTY (100 µg/ml ampicillin, 0.1 % glucose). The masterplate was conserved by adding 50 µl of
2xTY, 45 % glycerol to each well and stored at -20 °C. The induction plate was incubated at 37 °C with
shaking for approximately 3 hours (until an OD600 nm of 0.8 – 1.0 was reached) and production of
44
Kirstin A. Zettlitz
Methods
soluble scFv was induced by adding 25 µl of 2xTY (100 µg/ml ampicillin, 6 mM IPTG) and plates were
incubated overnight at 30 °C with shaking. The next day, after centrifugation, 50 µl of supernatant
from each well of the induction plate (in 100 µl 2 % MPBS) was transferred to the corresponding well
of an ELISA plates (coated with antigen huTNFR1-Fc or moTNFR1-Fc at 0.5 µg/ml in PBS overnight at
4 °C). Detection was performed using anti-His6-tag antibody-HRP (Santa Cruz) 1/1000 in 2 % MPBS as
described in 2.2.9. Positive clones were used to inoculate 5 ml overnight cultures (LB, 100 µg/ml
amp, 1 % glc) for mini-prep.
2.2.3
Coupling peptides to carrier proteins
Peptides were coupled to the carrier protein BSA using glutaraldehyde. A solution of 5 mg/ml of the
synthetic peptides was prepared in PBS and the carrier protein was added at the correct molar ratio
(approximately 1 mole of peptide per 50 aa of the carrier) to a total volume of 2 ml. An equal volume
of a prepared solution of 0.2 % glutaraldehyde in PBS was slowly added to the peptide/protein
solution with constant agitation and incubated for 1 hour at RT. Glycine (1 M stock solution) was
added to a final concentration of 200 mM and incubated with stirring for 1 hour at RT. The peptideprotein conjugate was separated from the peptide by dialysis against PBS.
2.2.4
Biotinylation of antigen
For a 2 mg/ml protein sample (TNFR1-Fc) a 20-fold molar excess of Sulfo-NHS-SS-Biotin was used and
incubated with the protein on ice for 2 hours. The non-reacted Sulfo-NHS-SS-Biotin was removed by
dialysis. Biotinylation efficiency was checked by using Dynabeads and both supernatant and bead
fraction were analyzed by Western Blot using Streptavidin-HRP (1/5000) for detection.
2.2.5
Protein production
2.2.5.1 Periplasmic protein expression in E. coli TG1 (scFv, Hsp70-SBD, AlF-fusion)
For soluble expression, the scFv fragments in pAB1 were transformed into E. coli TG1 and expression
was induced with 1 mM IPTG. The protein was purified from the bacterial periplasm and purified by
immobilized metal ion chromatography IMAC as described in 2.2.6.1. Protein concentration was
determined photometrically (Nanodrop) and calculated using the calculated ε-value. Hsp70
fragments less than 100 amino acids were produced as fusion proteins with human serum albumin
domain 3 (AlF3: aa 382-582 of HSA). The DNA encoding AlF3 was cloned into vector pAB1 as NotIAlF3-His-EcoRI fragment resulting in pAB1-AlF3. The Hsp70 fragments were introduced upstream of
AlF3 via NcoI/NotI, the resulting plasmids were transformed into E. coli TG1 and expression was
induced with 1 mM IPTG. The protein was purified from the bacterial periplasm and purified by
immobilized metal ion chromatography IMAC.
45
Kirstin A. Zettlitz
Methods
2.2.5.2 Protein expression in BL21-DE3 (Hsp70-fragments)
The expression plasmids (pET-28b) were transformed into chemically competent E. coli BL21-DE3. For
protein production 200 ml 2xTY (0.1 % glucose, 35 µg/ml kanamycin) were inoculated with 2 ml of an
overnight culture and incubated shaking at 37 °C until an OD600
nm
of 0.8 - 1.0 was reached.
Expression was induced with IPTG (final concentration 1 mM) and the culture was incubated
overnight at room temperature with shaking. Cells were harvested by centrifugation; the pellet was
resuspended in 5 ml lysis buffer (100 mM NaH2PO4, 10 mM Tris, 8 M urea) and stirred for 30 min.
After centrifugation for 30 min at 10000 x g the cleared lysate was transferred to a microcentrifuge
tube and stored at -20 °C. The substrate-binding domain (aa 383-548) of human Hsp70 (pET-28bHsp70-SBD) was produced in E. coli BL21-DE3 as described above and purified from the whole cell
extract by IMAC.
2.2.5.3 Production of IgG humex in CHO-K1
Stable transfected CHO-K1 cells (Lonza) were selected for their ability to grow in glutamine-free
medium by gradual removal of glutamine in the presence of GS inhibitor MSX. Supernatants were
screened for amount of secreted product by Western Blot and for functional product by ELISA.
Recombinant IgG1 was purified from cell culture supernatants by protein A chromatography. In brief,
proteins were precipitated from supernatant with ammonium sulfate (60% saturation), resuspended
in PBS, adjusted to pH 8.0 (by adding 1/10 volume of 1 M Tris-HCl pH 8.0) and loaded onto a protein
A-Sepharose CL-4B column (Sigma-Aldrich). Bound protein was eluted with 100 mM glycine, pH 3.0
and by adding 1/10 volume of 1 M Tris-HCl pH 8.0 the pH was brought back to neutral. Proteincontaining fractions were dialyzed against PBS.
2.2.5.4 Production of chimeric TNFR1-Fc in HEK293
HEK293 cells were transfected with plasmid DNA using lipofectamine (Invitrogen, Karlsruhe,
Germany) and stably transfected clones were selected in the presence of zeocin as described (Muller
et al. 2007). Cells were expanded in RPMI, 5 % FCS, 2 mM L-glutamine to 90 % confluence. For
protein production, the medium was substituted with Opti-MEM I (Invitrogen, Karlsruhe, Germany)
and supernatant was collected every 3-4 days.
2.2.6
Protein purification
2.2.6.1 Immobilized Ni+ affinity chromatography, IMAC
Recombinant scFv proteins were purified by binding via their C-terminal histidin-tag to nickelnitrilotriacetic acid-agarose beads (Ni-NTA). A column of 0.5 - 1 ml Ni-NTA beads was equilibrated
with 10 - 20 ml PBS and the periplasmatic preparation was loaded. Unbound proteins were washed
46
Kirstin A. Zettlitz
Methods
off the column with 50 ml IMAC wash buffer containing 20 mM imidazole. For elution IMAC elution
buffer (200 mM imidazole) was added and fractions of 500 µl were collected. The elution fractions
were tested for protein content by Bradford test. According to the Bradford test elution fractions
were pooled to peak and side fraction and dialyzed over night against PBS. Purity of the fractions was
analysed by SDS-PAGE by comparison with crude extract, flow through and wash fraction.
2.2.6.2 Protein A affinity chromatography
Proteins containing a human Fc-part were purified from cell culture supernatant by protein A
chromatography. In brief, supernatants were adjusted to pH 8 by adding 1/10 volume of 1 M TrisHCl
pH 8.0 and loaded onto a protein A-sepharose CL-4B column (Sigma, Taufkirchen, Germany).
Unbound protein was removed by rinsing the column with 10 column volumes (CV) of 100 mM Tris
pH 8.0 followed by 10 CV of 10 mM Tris pH 8.0. Bound protein was eluted with 100 mM glycine pH
3.0, neutralized by adding 1/10 volume 1 M TrisHCl pH 8.0 and protein containing fractions were
dialyzed against PBS. Protein concentrations were determined photometrically and purity was
analyzed by SDS-PAGE and immunoblotting using an HRP-conjugated anti IgG (Fc specific) antibody.
2.2.7
Protein characterization
Size exclusion chromatography (SEC) was performed by HPLC using a BioSep-Sec-3000 column
(Phenomenex, Torrance) or a BioSuite™ 250, 5 µm HR SEC (Waters GmbH, Eschborn, Germany) and
PBS as mobile phase at a flow rate of 0.5 ml/min. The following standard proteins were used:
thryroglobulin (669 kDa), apoferritin (443 kDa), β-amylase (200 kDa), bovine serum albumin (67 kDa),
carbonic anhydrase (29 kDa), cytochrome c (12.4 kDa).
2.2.7.1 SDS-PAGE, Immunoblot and Western Blot analysis
SDS-PAGE analysis was performed according to Laemmli using separation gels of 8 – 15 % (Laemmli
1970).
For immunoblot analysis, cleared cell lysates were separated by SDS-PAGE and blotted onto a
nitrocellulose membrane (semidry blot). The membrane was blocked with 5 % MPBS, 0.1 % Tween 20
for 30 min at room temperature. Primary antibodies were added to the membrane in PBS, 0.3 % BSA,
0.02 % azide at a concentration of 5 µg/ml for 2 h at room temperature. After washing, bound
antibodies were detected with HRP-conjugated anti-human or anti-mouse secondary antibodies.
Membranes were developed with ECL substrate solution for 1 min.
For Western Blot analysis purified proteins were separated by SDS-PAGE and blotted onto a
nitrocellulose membrane (semidry blot) as described for immunoblot analysis. Detection was
performed with appropriate HRP-conjugated antibodies.
47
Kirstin A. Zettlitz
Methods
2.2.7.2 Thermal stability
The melting points of the scFv and IgG molecules were determined by dynamic light scattering using
a ZetaSizer Nano ZS (Malvern, Herrenberg, Germany). Approximately 100 µg of purified scFv was
diluted in PBS to a total volume of 1 ml and sterile-filtered into a quartz cuvette. Dynamic laser light
scattering intensity (kcps) was measured while the temperature was increased in 1 °C intervals from
35 to 65 °C with 2 min equilibration for each temperature step. The melting point was defined as the
temperature at which the light scattering intensity dramatically increased.
2.2.7.3 Deglycosylation
Antibodies (IgG tumex, IgG humex; 10 µg in PBS) were denatured at 95 °C for 5 min and subsequently
incubated with 2 units N-glycosidase F (Roche, Mannheim, Germany) in a volume of 30 µl at 37 °C
overnight. Subsequently, untreated and deglycosylated proteins were analyzed by SDS-PAGE and
stained with Coomassie.
2.2.8
Affinity measurements
All experiments were carried out with an Attana A100 C-Fast system. Binding experiments were
performed in PBS 0.005 % Tween 20 at a flow rate of 25 to 35 µl/min and temperature was
controlled at 20 °C. The antigen was immobilized on an Attana carboxyl sensor chip by amine
coupling at a concentration of 50 µg/ml according to the manufacturer’s protocol, resulting in a
frequency shift of 200 Hz. Varying concentrations of the antibodies from 3.9 to 250 nM were
analyzed for binding. The chip was regenerated with 10 mM glycine-HCl, pH 3.0. Buffer injections
were performed prior to each sample injection to use as a reference in Attester Evaluation.
Additionally, Attana biotin sensor chips were used together with a Mouse-IgG capture kit and a
Human-IgG capture kit, respectively, to capture the IgG molecules at 5 µg/ml followed by injections
of varying concentrations of antigen from 3 to 100 nM. Data were collected by Attester 3.0 (Version
3.1.1.8, Attana, Stockholm, Sweden) and analyzed by ClampXP (Myszka and Morton 1998). A mass
transport model (Myszka 1997) was fitted to the data.
2.2.9
ELISA
Antigen was coated onto polystyrene mictrotiter plates (Greiner Bio-One, Frickenhausen, Germany)
at different concentrations (Hsp70-SBD: 3 µg/ml; TNFR1-Fc: 0.5 – 1 µg/ml) in PBS overnight at 4 °C.
Remaining binding sites were blocked with PBS, 2 % skimmed milk powder (MPBS) for 2 h at room
temperature. Primary antibodies were added in 2 % MPBS at the indicated concentrations and
incubated for 1 h at room temperature. After washing, bound monoclonal antibodies were detected
with horseradish peroxidase-conjugated anti-human, anti-mouse, or anti-His secondary antibodies
diluted in 2 % MPBS. Detection was performed using TMB substrate (1 mg/ml TMB, sodium acetate
48
Kirstin A. Zettlitz
Methods
buffer, pH 6.0, 0.006 % H2O2). The reaction was stopped with 50 µl of 1 M H2SO4. Absorbance was
measured at 450 nm in an ELISA reader (Tecan infinite M200).
2.2.10 Flow cytometry
Binding to TNFR1-Fas or TNFR2-Fas transfected MEF cells was analyzed by flow cytometry. Cells (2 x
105) were incubated with dilution series of antibodies for 4 h at 4 °C (Benedict et al. 1997). Cells were
then washed with PBS and bound antibodies were detected with PE-labeled goat anti-mouse or antihuman antibody. Cells were analyzed by flow cytometry (Cytomics FC 500, Beckmann-Coulter,
Krefeld, Germany). Data were evaluated with the program WinMDI, version 2.9, and fitted with
GraphPrism software (La Jolla, USA) from 3 independent binding curves.
2.2.11 Cytotoxicity
Kym-1 cells (1.5 x 104 cells/100 µl) were grown in 96-well plates over night. A constant amount of
human soluble TNF (1.25 ng/ml in medium) was applied after preincubation with antibodies in
triplicates (concentrations as indicated in the figures) in medium for 1 h. After 7 h cells were stained
by crystal violet (20 % methanol, 0.5 % crystal violet) for 20 min. The wells were washed with H2O
and air-dried. The dye was resolved with methanol for 20 min and optical density at 550 nm was
determined (Tecan infinite M200).
2.2.12 IL-6 and IL-8 assays
HT1080 cells (2.0 x 105 cells/100 µl) were grown in 96-well plates over night. The next day, the
medium was exchanged to remove constitutively produced IL-8 and the cells were incubated in
duplicates together with serial dilution of human soluble TNF for additional 18 h. Induction of IL-8
production and secretion into the culture supernatant was determined by an IL-8-Sandwich ELISA
(ImmunoTools, Friesoythe, Germany) according to the manufacturer’s protocol. In addition, cells
were incubated with serial dilutions of antibodies in presence (constant TNF 1 ng/ml) and absence of
TNF and analyzed for IL-8 secretion after 18 h of incubation. In the same way, we analyzed the
inhibitory effects of the antibodies on TNF-mediated secretion of IL-6 from HeLa cells using an IL-6
sandwich ELISA (ImmunoTools, Friesoythe, Germany) according to the manufacturer’s protocol.
2.2.13 Preparation of peripheral blood mononuclear cells (PBMC)
Buffy coat (leukapheresis) from a healthy human donor was diluted 1:4 in RPMI 1640, layered onto a
LSM 1077 Ficoll/ Hypaque gradient (PAA, Cölbe, Germany), and centrifuged for 20 min at 670 x g
(without deceleration) at room temperature. The PBMC fraction was aspirated and washed twice
with medium, before resuspending in 10 % Me2SO, 90 % fetal bovine serum and storing at -80 °C.
49
Kirstin A. Zettlitz
Methods
2.2.14 Isolation of human polymorphonuclear cells (PMN) and flow cytometry assay for
oxidative burst using dihydrorhodamine (DHR)
PMN were freshly prepared from 20 ml blood and purified using Polymorphprep™ (prod.no.
1114683, Axis-Shield, Oslo, Norway). Whole undiluted blood from a healthy human donor was
layered onto 5 ml Polymorphprep (1:1 vol with blood) and centrifuged for 30 min at 450 – 500 x g at
room temperature. After the separation both the top band containing only lymphocytes and
monocytes and the bottom band containing all of the PMNs were aspirated, pooled and washed two
times with medium. Marginal contamination of the PMN by erythrocytes was observed. Cell numbers
were determined and cells were adjusted to 2 x 106 cells/ml. Cells (200 000) were directly incubated
with TNF, ATROSAB or human control IgG at concentration of 1, 10, and 100 nM for 1 h. As positive
control cells were incubated with phorbolester (PMA). Degranulation was determined by adding
dihydrorhodamine (DHR) and subsequent FACS analysis gating the granulocyte population.
50
Kirstin A. Zettlitz
Results
3 Results
3.1 Hsp70-specific antibodies for cancer therapy
3.1.1
Chimerization of cmHsp70.1 (IgG tumex)
As a first approach towards a humanized antibody, a mouse-human chimeric antibody was
constructed (Zettlitz 2007). The sequences encoding the variable domains of monoclonal antibody
cmHsp70.1 were obtained by PCR amplification from cDNA from hybridoma cells using degenerated
primer annealing at the 5’ and 3’ region of the variable domains (Zettlitz et al. 2010). The VH and VL
domains were assembled into the scFv format (scFv tumex) and produced in E. coli. Additionally, an
IgG-like scFv-Fc fusion protein containing two binding sites per molecule and the human γ1 heavy
chain Fc region including the hinge region and a C-terminal hexahistidyl-tag was generated and
produced in HEK293 cells.
A chimeric IgG1 tumex was generated by fusing the VH and VL domains of cmHsp70.1 to the constant
regions of human γ1 heavy chain and the human Cλ domain, respectively (Zettlitz 2007). In this work
both heavy chain and light chain genes were cloned into a bicistronic expression vector (pEE14.4,
Lonza Biologics, Berkshire, UK) and transfected into CHO-K1 cells (Lonza Biologics, Berkshire, UK).
Transfected cells were selected for their ability to grow in glutamine-free medium by gradual
removal of glutamine in the presence of the glutamine-synthetase inhibitor L-methionine
sulphoximine (MSX). Approximately 300 positive clones were screened by ELISA for expression of
Hsp70-binding IgG and thereof 20 clones were expanded and screened by Western Blot for the
amount of IgG produced. 9 - 12 mg chimeric IgG tumex per liter of cell culture supernatant of the
best producers were purified by protein A chromatography. Purity and correct assembly of the IgG
molecules was confirmed by SDS-PAGE and size exclusion chromatography (SEC) analysis.
The SDS-PAGE analysis showed the characteristic heavy chain (calculated Mr: 48.9 kDa) and light
chain (calculated Mr: 22.7 kDa) bands under reducing conditions (Figure 3-1a) and a single band
corresponding to the assembled IgG (calculated Mr: 143.1 kDa without glycosylation) under nonreducing conditions. The apparent molecular mass (greater than 250 kDa) differed from the
calculated, this may be due to N-glycosylation of the heavy chain and the branched structure of the
IgG molecule. In immunoblot analysis the identity of purified IgG tumex was confirmed by detection
with an anti-human IgG (whole molecule) antibody that recognized both heavy and light chain under
reducing conditions and by an anti-human light chain (λ-specific) antibody that recognized the
assembled IgG under non-reducing conditions (Figure 3-1b). Size exclusion chromatography showed
51
Kirstin A. Zettlitz
Results
that IgG tumex eluted as a single peak again with a higher apparent molecular mass as calculated
(greater than 200 kDa) (Figure 3-1c). The parental mouse IgG1 cmHsp70.1 in comparison showed a
single peak at the same retention time corresponding to an apparent molecular mass of
approximately 200 kDa. A shoulder at 67 kDa represents the contained albumin (HSA).
Figure 3-1: Characterization of chimeric IgG tumex. a) SDS-PAGE (12%) analysis of purified IgG tumex (10 µg/lane) under
reducing (1) and non-reducing (2) conditions (Coomassie staining). b) Immunoblot analysis of purified IgG tumex (1
µg/lane). SDS-PAGE under reducing (1) and non-reducing (2) conditions; the heavy and light chain were detected by HRPconjugated anti-human IgG (whole molecule) antibody (1) and by HRP-conjugated anti-humanLC (λ-spec.) antibody (2). c)
SEC analysis of purified IgG tumex. d) SEC analysis of cmHsp70.1 (containing HSA).
The purified IgG tumex recognized recombinant human Hsp70 (not shown) and recombinant
substrate binding domain of human Hsp70 (rhHsp70-SBD) in ELISA with a similar sensitivity
(EC50=1.721 nM) as the parental antibody cmHsp70.1 (EC50=1.703 nM) (Figure 3-2). The results for
the chimeric IgG tumex confirmed that the correct mouse variable regions have been cloned and
retained the antigen binding properties of the parental mouse monoclonal antibody cmHsp70.1. In
addition the chimeric IgG is a valuable positive control for evaluating a prospective humanized
antibody.
Figure 3-2: Binding of IgG tumex and cmHsp70.1 to recombinant human Hsp70-SBD in ELISA. Binding to Hsp70-SBD
coated plates was detected by HRP-conjugated antibodies (anti-human IgG 1/2000 and anti-mouse IgG 1/2000,
respectively).
52
Kirstin A. Zettlitz
3.1.2
Results
Humanization of cmHsp70.1
Humanization by CDR-grafting is now a well established technique to reduce the immunogenicity of
xenogeneic (here mouse, commonly rodent) monoclonal antibodies and to improve their activation
of the human immune system. In order to humanize an antibody with retained antigen specificity
and binding affinity, the design of the humanized antibody is the critical step.
3.1.2.1 Analysis of mouse variable regions
Web Antibody Modelling (WAM, http://antibody.bath.ac.uk/) and Dr. Andrew C.R. Martin's Group
(http://www.bioinf.org.uk/) were used to number and analyse the variable regions derived from
hybridoma cmHsp70.1 (Figure 3-3, Figure 3-4). Sequences were numbered using the Kabat
numbering scheme. Framework regions (FR) and complementarity determining regions (CDR) were
defined based on the contact definition, this definition is residues which take part in interactions
with antigen and are part of the canonical sequence for loop structure (MacCallum et al. 1996), also
covering the different definitions provided in the literature (Kabat, Cothia, AbM) (Martin 2001)
(Figure 3-3). Canonical class alignment results of the CDRs are shown in Table 3-1.
Table 3-1: Canonical class alignment results using auto-generated SDR templates.
CDR
class
CDR
class
L1
7/14B
H1
?
similar to class 1/10A, but: H20=F (allows:
LIMV), H33=S (allows: YAWGTLV)
L2
1/7A
L3
?
H2
1/9A
similar to class ?/9D, but:
L96=L (allows: W)
Some unusual sequence features were identified in the VH domain, namely H20 F (occurs in 0.251 %
of heavy chains), H68 N (occurs in 0.451 % of heavy chains) which constitutes a potential Nglycosylation site and H101 H (occurs in 0.752 % of heavy chains). Also residues that are located at
the VH/VL interface and in the Vernier zone underlying the CDRs were identified (Foote and Winter
1992) (Figure 3-3).
53
Kirstin A. Zettlitz
Results
VH tumex
FR1
CDR1
FR2
CDR2
1
2
3
4
5
6
1234567890123456789012345 6789012345 67890123456 7890123456789012345
EVKLQESGPGLVAPSQSLSFTCTVS GFSLSRNSVH WVRQPPGKGLE WLGMIWGGGSTDYNSALKS
FR3
CDR3
FR4
7
8
9
10
11
67890123456789012abc345678901234 567890a12 34567890123
RLNISKDSSKSQVFLKMNSLQTDDTAMYFCAR NGGYDVFHY WGQGTTVTVSS
VL tumex
FR1
CDR1
FR2
CDR2
1
2
3
4
5
1234567891234567890123 4567abc890123456 789012345 67890123456
QAVVTQESALTTSPGETVTLTC RSSTGAVTTSNYANWV QEKPDHLFT GLIGGTNNRAP
FR3
CDR3
FR4
6
7
8
9
10
78901234567890123456789012345678 901234567 890123456a7
GVPARFSGSLIGDKAALTITGAQTEDEAIYFC ALWYSNHLV FGGGTKLTVLG
CDR-definition: Contact
AbM
Kabat
Chothia
Figure 3-3: Sequence analysis of mouse variable domains. Residues were numbered according to the Kabat numbering
scheme. Unusual sequence features are indicated in red. Potential N-glycosylation site is depicted in a grey box. Vernier
zone residues are indicated in green. Residues located at the VH/VL interface are underlined. CDR definitions according to
the different definitions are marked by colored lines.
3.1.2.2 Design of reshaped human antibody
A similarity search to human germline gene sequences was performed using V-BASE
(http://vbase.mrc-cpe.cam.ac.uk/) and IgBlast (http://www.ncbi.nlm.nih.gov/igblast/) to select the
human light and heavy chain variable regions to serve as template for CDR-grafting. For the VH the
most homologous human germline genes were of distinct canonical classes, therefore an additional
similarity search was performed for each FR separately. The selected human variable regions were
analysed for percent similarity with the mouse variable regions, confirmation of the identical
canonical CDR structures for the parental and humanized variable sequences (Martin and Thornton
1996) and the assigned humanness z-score (Figure 3-6: Z-score values of variable regions.)
(Abhinandan and Martin 2007).
54
Kirstin A. Zettlitz
Results
The most appropriate human sequences (the most similar variable region and the one with the
highest z-score, humanness) were chosen for virtual CDR-grafting and superimposed modeling of the
reshaped Fv’s (Figure 3-4). Human germline sequence VL 3-2 (IGLV7-43 IMGT, DPL18) and VH 3-66
(IGHV3-66 IMGT, DP-86) were chosen subjectively for retaining the loop structure of the grafted
CDRs. A humanized version of murine cmHsp70.1 (designated humex, Figure 3-4, Figure 3-5) was
generated by CDR grafting onto these human germline sequences.
Figure 3-4: Superimposed model structure of Fv tumex (green) and Fv humex (blue) backbone. Side view of the two
model structures. Cysteines are depicted as yellow spheres. The darker regions show the grafted CDR regions. The model
was generated with Web Antibody Modeling (WAM, http://antibody.bath.ac.uk/) and visualized with PyMol
(http://www.pymol.org).
55
Kirstin A. Zettlitz
Results
Figure 3-5: Sequence alignment. Residues identical to the parental mouse sequences are shown as dots. Residues were
numbered according to the Kabat numbering scheme. a) Alignment of the VH sequence isolated from cmHsp70.1
hybridoma (VH tumex) with the closest mouse germline gene (VHQ52.a19.61) as well as the human germline sequence
(3-66) used as scaffold for humanization. The humanized VH sequence (VH humex) is also shown. b) Alignment of the VL
sequence isolated from cmHsp70.1 hybridoma (VL tumex) with the closest mouse germline gene (VL1) as well as the
human germline sequence (3-2) used as scaffold for humanization. The humanized VL sequence (VL humex) is also
shown.
The calculated z-scores for humanness (Abhinandan and Martin 2007) (Figure 3-6) were increased
after humanization from -1.268 for VH tumex to 0.899 for VH humex (acceptor human germline
sequence: 2.178) and from -3.471 for VL tumex to -2.142 for VL humex (acceptor human germline
sequence: -2.077).
a)
b)
Figure 3-6: Z-score values of variable regions. The Z-score of the query-sequence is indicated by a red line. The
distribution of human germline sequences is shown in magenta, the distribution of mouse germline sequences is shown
in blue. a) Comparison of IGHV of the original mouse VH tumex, the chosen human germline sequence VH3-66 and the
reshaped humanized VH humex. b) Comparison of IGLV of the original mouse VL tumex, the chosen human germline
sequence VL3-2 and the reshaped humanized VL humex.
3.1.2.3 ScFv humex
Codon optimized VH and VL domains (GeneArt, Regensburg, Germany) were assembled into the scFv
format (scFv humex) and produced in E. coli with yields of 1 ± 0.3 mg per liter culture, which is a 5fold
improvement compared to scFv tumex (0.2 ± 0.1 mg per liter culture) see Table 3-2.
Table 3-2: Production yields.
protein
cell system
purification
yield [mg/L supernatant]
scFv tumex
E. coli TG1
IMAC
0.1 – 0.4
scFv-Fc tumex
HEK 293
IMAC
4.1
IgG1 tumex
CHO-K1 Lonza GS system, (pEE12.4)
protein A
0.7
protein A
9 - 12
scFv humex
E. coli TG1
IMAC
0.7 – 1.3
IgG1 humex
protein A
> 20
56
Kirstin A. Zettlitz
Results
SDS-PAGE analysis revealed a single band with an apparent molecular mass of about 28 kDa
(calculated Mr 27.9 kDa) both under reducing and non-reducing conditions (Figure 3-7a). Immunoblot
analysis confirmed the identity of scFv humex (Figure 3-7b). Size exclusion chromatography showed
that scFv humex eluted as a single peak, corresponding to an apparent molecular mass of
approximately 28 kDa (Figure 3-7c). The purified scFv humex recognized recombinant human Hsp70SBD in ELISA (Figure 3-7d) confirming that humanization was successful and antigen specificity was
retained.
Figure 3-7: Characterization of humanized scFv humex. a) SDS-PAGE analysis of purified scFv humex (2 µg/lane) under
reducing (1) and non-reducing (2) conditions (Coomassie staining). b) Immunoblot analysis of purified scFv humex (1
µg/lane) under reducing (1) and non-reducing (2) conditions detected by HRP-conjugated anti-His antibody. c) Size
exclusion chromatography of scFv humex. d) Binding of scFv humex to recombinant human Hsp70-SBD in ELISA. Binding
was detected by HRP-conjugated anti-His antibody.
Humanization also resulted in improved thermal stability, which was determined by measuring the
dynamic light scattering intensity (kcps) while the temperature was increased in 1 °C intervals
(ZetaSizer Nano ZS, Malvern Instruments, Herrenberg, Germany) (Seitter 2010; Zettlitz et al. 2010).
As the protein aggregated the light scattering intensity increased dramatically and this temperature
was defined as melting point. The melting point for scFv tumex was determined to be approximately
47 °C while an increased melting point of approximately 58 °C was measured for scFv humex (Figure
3-8).
Figure 3-8: Analysis of the melting point of scFv tumex and scFv humex by dynamic light scattering. Dynamic light
scattering intensity (kcps) was measured while temperature was increased in 1 °C intervals.
57
Kirstin A. Zettlitz
Results
3.1.2.4 IgG humex
The humanized VH and VL encoding regions were used to generate a humanized IgG1 (IgG humex) in
the same way as described for chimeric IgG tumex, but using a modified Igκ-leader allowing correct
N-terminal processing. Stable transfected CHO-K1 cells were screened by ELISA for expression of
functional (Hsp70-binding) IgG and thereof 10 cell lines were expanded and screened by Western
Blot for the amount of IgG produced. More than 23 mg humanized IgG humex per liter of cell culture
supernatant were purified by protein A chromatography (Table 3-2). Purity and correct assembly of
IgG humex was confirmed by SDS-PAGE analysis and by size exclusion chromatography. SDS-PAGE
analysis showed under reducing conditions two bands with an apparent molecular mass of 53 kDa
and 25 kDa, corresponding to the heavy and light chain of IgG humex (calculated molecular masses:
48.8 kDa and 22.6 kDa, respectively). Under non-reducing conditions a single band migrating with an
apparent molecular mass of more than 250 kDa was visible, corresponding to the assembled IgG
(calculated Mr: 142.8 kDa without glycosylation) (Figure 3-9a). The difference between calculated and
apparent molecular mass may be caused by N-glycosylation of the heavy chain and the branched
structure of IgG humex. In immunoblot analysis the identity of purified IgG humex was confirmed by
detection with an anti-human light chain (λ-specific) antibody that recognized the lambda light chain
under reducing conditions and by an anti-human IgG (Fc-specific) antibody that recognized the heavy
chain under reducing conditions and the assembled IgG under non-reducing conditions (Figure 3-9b).
Size exclusion chromatography showed that IgG humex eluted as a single peak of more than 200 kDa,
again with a higher apparent weight as calculated (Figure 3-9c). In ELISA the purified IgG humex
recognized rhHsp70 (not shown) and rhHsp70-SBD in a dose-dependent manner (Figure 3-9d).
Figure 3-9: Characterization of humanized IgG humex. a) SDS-PAGE analysis of purified IgG humex (3 µg/lane) under
reducing (1) and non-reducing (2) conditions (Coomassie staining). b) Immunoblot analysis of purified IgG humex (0.5
µg/lane) under reducing (1,2) and non-reducing conditions (3), detected by HRP-conjugated anti-human LC (λ-spec.)
antibody (1) and anti-human IgG (Fc-spec.) antibody (2,3), respectively. c) Size exclusion chromatography of purified IgG
humex. d) Binding of IgG humex to rhHsp70-SBD in ELISA. Bound antibody was detected by HRP-conjugated anti-human
IgG (whole molecule) antibody.
58
Kirstin A. Zettlitz
3.1.3
Results
Antibody glycosylation
Incubation of purified IgG tumex and IgG humex (15 µg each) with PNGase F confirmed Nglycosylation of the heavy chain of both antibodies. Before treatment, the heavy chain of IgG tumex
migrated with an apparent molecular mass of approximately 56 kDa, which was reduced to 52 kDa
after PNGase treatment. The heavy chain of IgG humex migrated with an apparent molecular mass of
approximately 53 kDa before treatment and was reduced to 51 kDa after PNGase treatment (Figure
3-10). The increased reduction of the molecular mass by 2 kDa of IgG tumex heavy chain after
deglycosylation as compared to the IgG humex heavy chain indicates that the additional sequon in
the variable heavy chain domain of IgG tumex is indeed N-glycosylated. The light chain was not
affected by the treatment with PNGase F (28 kDa for IgG tumex and 26 kDa for IgG humex). The
differences in the molecular mass between the chimeric and the humanized antibody were caused by
different N-terminal processing due to the used Igκ-leader sequences.
Figure 3-10: Antibody glycosylation. IgG tumex (1,2) and IgG humex (3,4) were incubated with PNGase F (2,4) and
subsequently analyzed by SDS-PAGE analysis under reducing conditions (Coomassie staining). An increased mobility of
the treated samples indicates that both antibodies are N-glycosylated.
3.1.4
Affinity measurement
The affinities of cmHsp70.1, IgG tumex, IgG humex and scFv humex were determined by quartz
crystal microbalance (QCM) measurements using an Attana A100® C-Fast system. The interaction of
antigen and antibody was examined in two settings with the antigen (rhHsp70-SBD) immobilized or
the antibody captured on the sensor chip, respectively. These two settings allow estimation of avidity
effects which would affect the measured apparent affinity. The affinities of mouse cmHsp70.1,
chimeric IgG tumex, humanized IgG humex and scFv humex for immobilized rhHsp70-SBD were
determined to be 2.8 nM, 1 nM, 1.5 nM, and 6 nM, respectively (Table 3-3, Figure 3-11 1a-d). In the
second set of experiments using captured antibodies and soluble rhHsp70-SBD all IgG’s showed
similar kinetics with affinities of 6 nM, 3.3 nM and 4.2 nM for cmHsp70.1, IgG tumex and IgG humex,
respectively (Table 3-3, Figure 3-11 2a-c). In both settings, all three antibodies showed similar
59
Kirstin A. Zettlitz
Results
kinetics with affinities to Hsp70-SBD in the low nanomolar range, thus confirming that humanization
had not affected affinity.
Table 3-3: Summary of kinetic data from QCM measurements of Hsp70-specific antibodies.
construct
method
kon (M-1s-1)
cm Hsp70.1
immobilized Ag
2.5 x 10 ± 4.2 x 10
captured Ab
IgG tumex
IgG humex
scFv humex
immobilized Ag
koff (s-1)
5
4
5
3
5
4
5
2
5.2 x 10 ± 3.4 x 10
5
3
3.3 x 10 ± 6.1 x 10
5
2
4.7 x 10 ± 3.9 x 10
5
3
8.2 x 10 ± 4.7 x 10
2.5 x 10 ± 6.1 x 10
3.6 x 10 ± 3.5 x 10
captured Ab
1.6 x 10 ± 5.2 x 10
immobilized Ag
2.1 x 10 ± 8.4 x 10
captured Ab
4.3 x 10 ± 4.9 x 10
immobilized Ag
1.4 x 10 ± 6.7 x 10
KD (nM)
Rmax (Hz)
-4
-5
2.8
10
-3
-5
6.0
168
-4
-5
1.0
23
-4
-6
3.3
105
-4
-6
1.5
37
-4
-6
4.2
181
-4
-6
6.0
40
6.9 x 10 ± 6.3 x 10
1.5 x 10 ± 1.2 x 10
3.5 x 10 ± 1.5 x 10
Figure 3-11: Affinity determination of Hsp70-specific antibodies by quartz crystal microbalance (QCM) measurements. 1)
Binding of bivalent (a-c) and monovalent (d) Hsp70-specific antibodies to immobilized rhHsp70-SBD. 2) Binding of soluble
rhHsp70-SBD to captured mouse (a) and human (b,c) IgG.
3.1.5
Epitope mapping of Hsp70-specific antibodies
Competition ELISA with parental mouse antibody cmHsp70.1 was performed to confirm that the
recombinant antibodies retained original epitope specificity. Binding of cmHsp70.1 to immobilized
antigen (rhHsp70-SBD) was inhibited by increasing concentrations of IgG tumex or IgG humex in a
dose-dependent manner. No inhibition was observed with an irrelevant chimeric IgG (IgG CEA,
(Zettlitz 2007; Stork et al. 2008)). This result indicated that both recombinant antibodies bind the
same epitope as the parental antibody (Figure 3-12a). Binding of both the chimeric IgG tumex and
the humanized IgG humex to heat shock-induced Hsp70 of human and murine cell lines was shown in
immunoblotting experiments (Figure 3-12b).
60
Kirstin A. Zettlitz
Results
Figure 3-12: Antigen binding. a) Competition ELISA. cmHsp70.1 (constant 10 nM) was incubated with increasing amounts
of IgG tumex, IgG humex and IgG CEA, respectively. Remaining antigen binding activity of cmHsp70.1 was determined by
ELISA with immobilized antigen (rhHsp70-SBD). b) Immunoblot experiments for binding of cmHsp70.1, IgG tumex and IgG
+
humex to heat shock-induced extracts of human Colo and murine B16 cells, respectively. Purified recombinant human
Hsp70 (rhHsp70) was included as positive control. In both experiments (a,b) bound antibodies were detected by HRPconjugated anti-human IgG and anti-mouse IgG antibodies, respectively.
To further analyze the epitope specificity of the Hsp70-specific antibodies, binding and blocking
experiments using synthetic TKD-peptide containing the proposed epitope (aa450-463) (Multhoff
2007) were performed. Synthetic TKD-peptide in 105-fold molar excess was not able to block binding
of cmHsp70.1 (Figure 3-13a) or IgG tumex (Figure 3-13b) to immobilized Hsp70 in ELISA. In this assay
soluble Hsp70 in 830-fold molar excess completely blocked binding of both antibodies. Binding of the
recombinant antibodies was also investigated using a TKD-fusion protein comprised of HSA with two
TKD-peptides fused N- and C-terminal, in immunoblot experiments no binding of cmHsp70.1 or scFvFc tumex could be observed (Figure 3-13c).
Figure 3-13: Blocking and binding experiments with synthetic TKD-peptide. Blocking of cmHsp70.1 (a) and IgG tumex (b)
binding to coated Hsp70 (100 ng/well) by soluble TKD-peptide (100.000-fold molar excess) and Hsp70 (830-fold molar
excess). c) Immunoblot analysis of binding of cmHsp70.1 and scFv-Fc tumex to recombinant Hsp70 (1) and TKD-HSA-TKD
(2).
Synthetic peptides containing the epitope sequence and adjacent amino acids were used to repeat
binding and blocking experiments by ELISA and Immunoblot analysis. None of the synthetic peptides
used in this experiments (Figure 3-14a) showed binding of Hsp70-specific antibodies in Immunoblot
(Figure 3-14b) or ELISA experiments or was able to block binding of these antibodies to Hsp70.
61
Kirstin A. Zettlitz
Results
Figure 3-14: a) Synthetic peptides comprising the proposed epitope sequence and adjacent amino acids. b) Dot blot
analysis of binding of Hsp70-specific antibodies (cmHsp70.1 and IgG tumex) to synthetic peptides blotted in indicated
concentrations onto a nitrocellulose-membrane. Bound antibody was detected by HRP-conjugated anti-mouse IgG or
anti-human IgG antibodies, respectively.
Coupling of the synthetic peptides to a carrier protein (BSA) was achieved by using glutaraldehyde
(Figure 3-15a), and was thought to support immobilization of the small peptides. Nevertheless,
binding to these BSA-coupled peptides did not occur neither in dot blot (Figure 3-15b) nor in ELISA
experiments (Figure 3-15c).
Figure 3-15: BSA-coupled peptides. a) Successful coupling of synthetic peptides to BSA. BSA-NGL-14 (2), BSA-NLL-17 (3)
and BSA-RFE-13 (4) analysed by SDS-PAGE under reducing conditions (3 µg/lane, Coomassie-staining). Uncoupled BSA
was included as control (1). b) Dot blot analysis of binding of cmHsp70.1 (1) and IgG tumex (2) to blotted BSA-coupled
peptides (10 µg/dot). Hsp70 (0.1 µg/dot) and BSA (10 µg/dot) were included as control. c) Binding of cmHsp70.1 and IgG
tumex to peptides and BSA-coupled peptides in ELISA. Hsp70 was included as control. Bound antibody was detected by
HRP-conjugated anti-mouse IgG or anti-human IgG antibodies.
These findings indicate that the proposed epitope region (aa 454-461, NLLGRFEL) (Botzler et al. 1998)
is not sufficient for antibody binding. In order to localize the entire epitope, overlapping Hsp70fragments were cloned and produced in E. coli. Fragments smaller than 100 amino acids were fused
to human serum albumin domain 3 (AlF3: HSA aa 382-582) to facilitate purification and SDS-PAGE
analysis. Binding of Hsp70-specific antibodies to the various fragments was analyzed by immunoblot
experiments. In these experiments all three antibodies (mouse cmHsp70.1, chimeric IgG tumex, and
humanized IgG humex) showed an identical binding pattern. The minimal region showing antibody
binding in this assay was located within aa 473 - 504 of the C-terminal substrate binding domain
(Figure 3-16). N- or C-terminal deletion of this region resulted in complete loss of binding (Figure
3-16, fragment 8, 16), indicating that the entire region of 32 amino acids was required.
62
Kirstin A. Zettlitz
Results
Figure 3-16: Epitope mapping. In the scheme of Hsp70-fragments antibody binding is indicated by green bars, Hsp70fragments that did not bind are depicted in red. The table shows position, length and immunoblot result of all used
Hsp70-fragments.
A structural model of the Hsp70-SBD (substrate binding domain, aa 383 - 548) displays the respective
amino acids in three beta strands close to each other (Figure 3-17b). This region is located adjacent
to a beta strand containing the TKD sequence. Sequence analysis of the identified sequence revealed
100 % identity to mouse Hsp70 (HS71B_mouse), while three substitutions are located in human
Hsc70 (HSP7C_human) (Figure 3-17c).
a)
b)
c)
Figure 3-17: Epitope mapping. a) Immunoblot experiment with fragment 18 (KITI, aa 473-504) fused to domain III of HSA
incubated with Hsp70-specific antibodies; HRP-conjugated anti-His-tag antibody as control. Recombinant scDb-AlF3 was
included as negative control. b) Structure of Hsp70-SBD showing the position of aa 473-504 and the adjacent TKD
sequence (light pink). Structures were visualized with PyMol (http://www.pymol.org). c) Sequence of human Hsp70 (aa
450-504) showing the identified epitope (underlined) of the Hsp70-specific antibodies. The previously proposed epitope
is depicted in light pink. Alignment of aa 473-504 of human Hsp70, human Hsc70 and mouse Hsp70.
63
Kirstin A. Zettlitz
Results
3.2 Antagonistic TNFR1-specific antibodies for therapy of inflammatory
diseases
3.2.1
Antibody constructs derived from humanized H398 (IZI-06.1)
The hybridoma producing monoclonal mouse antibody H398 (subtype IgG2a) was generated by
immunization of mice with purified TNF-receptor 1 (Thoma et al. 1990). This antagonistic antibody is
specific for human TNFR1 and neutralized a wide spectrum of TNF activities in vitro by competitive
inhibition of TNF binding to TNFR1. H398 shows no crossreactivity with TNF receptors from most
other species, therefore standard small animal models cannot be used for in vivo evaluation. As well,
it would be difficult to evaluate the therapeutic efficacy of H398 in clinical trials, because of the
mouse origin of the antibody, holding the risk of acute adverse effects and development of human
anti-mouse antibodies (HAMA) upon repeated treatment cycles. To overcome these limitations a
humanized version of H398, designated IZI-06.1, was generated by grafting all six complementaritydetermining regions (CDRs) onto human variable germline genes similar to H398 VH and VL sequences
(Kontermann et al. 2008). A bacterially produced Fab fragment of IZI-06.1 showed equal blocking
activity as the respective mouse Fab fragment. Thus, this humanized TNFR1-specific antibody holds
great promise for development of a therapeutic. A clinically applicable therapeutic requires an
appropriate format which shows good binding, pharmacokinetics and production. Based on IZI-06.1,
the humanized version of mouse mAb H398, different antibody constructs were generated.
3.2.1.1 IZI-06.1 scFv
The variable domains of IZI-06.1 were combined into the scFv format using vector pHENIS and
subcloned into vector pAB1 for production in E. coli TG1. Purified IZI-06.1 scFv was characterized by
SDS-PAGE analysis and size exclusion chromatography. A single band of approximately 29 kDa was
visible under both reducing and non-reducing conditions in SDS-PAGE analysis (calculated Mr: 29.4
kDa). In size exclusion chromatography IZI-06.1 scFv eluted as three distinct peaks one of which
corresponding to the monomer while the others indicated formation of dimers and trimers.
64
Kirstin A. Zettlitz
Results
Figure 3-18: Characterization of IZI-06.1 scFv. a) SDS-PAGE analysis of purified IZI-06.1 scFv (2 µg/lane) under reducing
(lane 1) and non-reducing conditions (lane 2), (Coomassie staining). b) Size exclusion chromatography of purified IZI-06.1
scFv (position of standard proteins is indicated).
3.2.1.2 IZI-06.1 Fab-HSA
In this construct, the Fd-fragment (VH-CH1 IZI-06.1) is fused to human serum albumin by a short
glycine-serine linker (GGGS). Using a bicistronic expression vector with the light chain (IZI-06.1 LC)
cloned upstream of the Fd-HSA, the Fab-HSA fusion protein was produced in CHO cells and purified
by Celonic. Purity and integrity were examined by SDS-PAGE analysis and size exclusion
chromatography (Figure 3-19). SDS-PAGE analysis under reducing conditions showed two bands
migrating with an apparent molecular mass of approximately 25 kDa and 90 kDa corresponding to
IZI-06.1 LC (calculated Mr: 23.9 kDa) and Fd-HSA (calculated Mr: 89.9 kDa), respectively (Figure 3-19a,
lane 3). Under non-reducing conditions the fully assembled IZI-06.1 Fab-HSA migrated as a band of
approximately 115 kDa (Figure 3-19a, lane 4). For comparison IZI-06.1 Fab and HSA were included.
Also some minor bands were visible, probably corresponding to impurities and degradation products.
In size exclusion chromatography (SEC) correct assembly of IZI-06.1 Fab-HSA was confirmed,
additionally a substantial amount of aggregates or impurities were observed (Figure 3-19b).
Figure 3-19: Characterization of IZI-06.1 Fab-HSA. a) SDS-PAGE analysis of purified proteins (2 µg/lane, Coomassie
staining) under reducing (1,2,3) and non-reducing (4,5,6) conditions. IZI-06.1 Fab-HSA (3,4); HSA (2,5) and IZI-06.1 Fab
(1,6) were included for comparison. b) Size exclusion chromatography of purified IZI-06.1 Fab-HSA (position of standard
proteins is indicated).
65
Kirstin A. Zettlitz
Results
3.2.1.3 IZI-06.1 scFv-HSA
The IZI-06.1 scFv was fused C-terminal to HSA by a short glycine-serine linker (GGGS). This singlechain peptide was produced in CHO cells and purified by Celonic. Purity and integrity were analyzed
by SDS-PAGE analysis and size exclusion chromatography (Figure 3-20a,b). Beside the monomeric
IZI-06.1 scFv-HSA of about 95 kDa, degraded as well as aggregated protein was visible in both
experiments although to a lesser extent than for the Fab-HSA.
Figure 3-20: Characterization of IZI-06.1 scFv-HSA. a) SDS-PAGE analysis of purified proteins (6 µg/lane, Coomassie
staining) under reducing (1,2,3) and non-reducing (4,5,6) conditions. IZI-06.1 scFv-HSA (3,4); HSA (2,5) and IZI-06.1 scFv
(1,6) were included for comparison. b) Size exclusion chromatography of purified IZI-06.1 scFv-HSA (position of standard
proteins is indicated).
3.2.1.4 IZI-06.1 IgG (ATROSAB)
The humanized anti-human TNFR1 antibody IZI-06.1 (Kontermann et al. 2008) was converted into a
human IgG1 using a heavy chain with abolished effector functions (IgGe3-Fc2, InvivoGen). The used
IgG1 is of the G1m1,17 allotype (IGHG1 allele name *01) and has substitutions of IgG2 residues at
positions 233 – 236 and IgG4 residues at positions 327, 330 and 331 (Figure 3-22).
Figure 3-21: Characterization of ATROSAB. a) SDS-PAGE analysis of purified ATROSAB (4 µg/lane, BioRad ready gel TrisHCl gel, 4-15% linear gradient, Coomassie staining) under non-reducing (1) and reducing conditions (2). b) Size exclusion
chromatography of purified ATROSAB (the position of standard proteins is indicated).
66
Kirstin A. Zettlitz
Results
This antibody (named ATROSAB) was produced in CHO cells and purified by protein A
chromatography (Celonic). Purity and integrity were confirmed by SDS-PAGE and size exclusion
chromatography. In SDS-PAGE analysis (4 - 15 % linear gradient) under non-reducing conditions a
band of approximately 160 kDa was visible corresponding to the fully assembled IgG ATROSAB.
Under reducing conditions two bands corresponding to the heavy (calculated Mr: 53 kDa) and light
chain (calculated Mr: 25 kDa) were observed (Figure 3-21a). In size exclusion chromatography,
purified ATROSAB eluted as a single peak with an apparent molecular mass of approximately 160 kDa
(Figure 3-21b).
a)
VL:
1
2
3
4
5
12345678901234567890123 4567abcde8901234 567890123456789 0123456
DIVMTQSPLSLPVTPGEPASISC RSSQSLLHSNGNTYLH WYLQKPGQSPQLLIY TVSNRFS
6
7
8
9
10
78901234567890123456789012345678 901234567 8901234567
GVPDRFSGSGSGTDFTLKISRVEAEDVGVYYC SQSTHVPYT FGGGTKVEIK
CK:
110
120
130
140
150
160
170
180
190
.
.
.
.
.
.
.
.
.
RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKV
200
210
.
.
YACEVTHQGLSSPVTKSFNRGEC
b)
VH:
1
2
3
4
5
6
123456789012345678901234567890 12345 67890123456789 012a3456789012345
QVQLVQSGAEVKKPGSSVKVSCKASGYTFT DFYIN WVRQVPGQGLEWIG EIYPYSGHAYYNEKFKA
7
8
9
10
11
67890123456789012abc345678901234 56781234567890123
RVTITADKSTSTAYMELSSLRSEDTAVYYCAR WDFLDYWGQGTTVTVSS
CH1:
120
130
140
150
160
170
180
190
.
.
.
.
.
.
.
.
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICN
200
210
.
.
VNHKPSNTKVDKKVEPKSC
Hinge:
230
.
DKTHTCPPCPAPPVAG
CH2:
240
250
260
270
280
290
300
310
320
.
.
.
.
.
.
.
.
.
PSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKC
330
340
.
.
KVSNKGLPSSIEKTISKAK
CH3:
350
360
370
380
390
400
410
420
.
.
.
.
.
.
.
.
GQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFS
430
440
.
.
CSVMHEALHNHYTQKSLSLSPGK
67
Kirstin A. Zettlitz
Results
Figure 3-22: Sequence of humanized IgG ATROSAB. Residues are numbered according to the Kabat numbering scheme. a)
Light chain of ATROSAB. b) Heavy chain of ATROSAB. The substituted residues of IgG2 and IgG4 are underlined. Lys214
determines the G1m17 allotype, whereas Asp356 and Leu358 determine the G1m1 allotype.
3.2.2
TNFR1-Fc fusion proteins
Fusion proteins composed of the extracellular domains of human TNFR1 (aa 29-211), rhesus TNFR1
(aa 27-209), and mouse TNFR1 (aa 30-212) fused to the hinge and Fc-region of human γ1 heavy chain
were produced in HEK293 cells and purified by protein A chromatography. Purified proteins were
analyzed by SDS-PAGE analysis and showed a single band with under reducing conditions
corresponding to the monomer (calculated Mr: 47 kDa without glycosylation). Under non-reducing
conditions the dimer is represented by a single band migrating with an apparent molecular weight
higher than 130 kDa (calculated Mr: 95 kDa without glycosylation) (Figure 3-23a). In size exclusion
chromatography human TNFR1-Fc eluted as a single peak (Figure 3-23b).
Figure 3-23: Characterization of TNFR1-Fc fusion proteins. a) SDS-PAGE analysis of purified human TNFR1-Fc (1,4), mouse
TNFR1-Fc (2,5) and rhesus TNFR1-Fc (3,6) (4 µg/lane, BioRad ready gel Tris-HCl gel, 4-15% linear gradient, Coomassie
staining) under reducing (1-3) and non-reducing (4-6) conditions. b) Size exclusion chromatography of purified human
TNFR1-Fc (the position of standard proteins is indicated).
3.2.3
Antigen specificity (species- and receptor-specificity)
Antigen binding of all IZI-06.1 constructs was confirmed by ELISA using human TNFR1-Fc coated
plates and HRP-conjugated secondary antibodies. IZI-06.1 scFv showed the strongest binding of the
monovalent constructs with an EC50 value of 2.6 nM. IZI-06.1 scFv-HSA and Fab-HSA bound slightly
weaker with EC50 values of 3.5 nM and 7.2 nM, respectively (Figure 3-24a). The bivalent IgG1
ATROSAB exhibited the strongest binding to recombinant human TNFR1-Fc (EC50 value: 0.22 nM),
similar to the binding of the parental antibody H398 (EC50 value: 0.1 nM) (Figure 3-24b). These results
confirmed that humanization and conversion into the different antibody formats did not affect
antigen binding.
68
Kirstin A. Zettlitz
Results
Figure 3-24: ELISA of IZI-06.1 constructs for binding to human TNFR1-Fc. a) ELISA of monovalent IZI-06.1 constructs.
Binding of scFv was detected by HRP-conjugated anti-His antibody and binding of HSA-fusion proteins was detected by
rabbit anti-HSA antibody and HRP-conjugated anti-rabbit antibody (n=3). b) ELISA of bivalent IgG ATROSAB and H398.
Binding of ATROSAB was detected by HRP-conjugated anti-huIgG (Fab-specific) antibody and binding of H398 was
detected by HRP-conjugated anti-mouse IgG antibody (n=3). Data were normalized to Y=0 becomes 0 % for all data set
and the largest value in each data set becomes 100 %. Data were fitted to the approximate equation of one site – specific
binding Y = Bmax * X/(EC50 + X), where Bmax is the extrapolated maximum specific binding and EC50 is the concentration
needed to achieve half-maximum binding at equilibrium.
The ability of ATROSAB and H398 to bind to antigen expressing cells and their selectivity for TNFR1
was confirmed by flow cytometric analysis using mouse embryonic fibroblasts (MEF) transfected with
fusion proteins comprised of the extracellular domain of TNFR1 and TNFR2, respectively, fused to the
intracellular domain of human Fas (MEF-TNFR1-Fas, MEF-TNFR2-Fas) (Krippner-Heidenreich et al.
2002). For both antibodies, binding was only seen with MEF-TNFR1-Fas cells but not with MEFTNFR2-Fas cells (Figure 3-25). Additionally, binding of TNFR1-specific antibodies to huTNFR1 was
confirmed by using Kym-1 cells (human rhabdomyosarcoma, expressing about 3000 TNFR1 molecules
per cell) in flow cytometry analysis.
Figure 3-25: Flow cytometry analysis of binding of ATROSAB and H398 to mouse embryonic fibroblasts (MEF) transfected
with human TNFR1-Fas or human TNFR2-Fas and to Kym-1 cells expressing human TNFR1. Cells alone are depicted in
grey; cells incubated with indicated antibodies are depicted as black line.
69
Kirstin A. Zettlitz
Results
Titration of antibody concentration showed that binding of ATROSAB to MEF-TNFR1-Fas cells was
comparable to that of H398 with EC50 values of approximately 0.09 nM for ATROSAB and 0.2 nM for
H398 (Figure 3-27).
Figure 3-26: Flow cytometry analysis of binding of ATROSAB and H398 to MEF-TNFR1-Fas cells. Titration of antibody
concentration (n=3), data were normalized to cells alone becomes 0 % and the largest value in each data set becomes
100 %. Data were fitted to the approximate equation MFI = MFI max * [IgG]/(EC50+[IgG]) using GraphPad Prism Software,
where MFI is the mean fluorescent intensity and MFImax is the fitted plateau value at high concentrations of IgG.
Species specificity was investigated by using recombinant TNFR1-Fc fusion proteins in ELISA
experiments. In this assay, binding of ATROSAB and H398 was seen for human and rhesus TNFR1-Fc,
but not for mouse TNFR1-Fc (Figure 3-27).
Figure 3-27: ELISA of binding of ATROSAB and H398 to purified human, rhesus and mouse TNFR1-Fc. Binding of an antihuman IgG (Fc-specific) antibody (a-huFc) was included as coating control.
3.2.4
Thermal stability
Thermal stability of purified ATROSAB was determined by dynamic light scattering (DLC) using 1 °C
intervals and 1 min for equilibration. ATROSAB showed a two-phase thermal denaturation with a first
transition around 50 °C and a second (major) transition at 81 °C. This indicates that ATROSAB is stable
up to a temperature of 46 °C (Figure 3-28a). Analysis of the IZI-06.1 scFv fragment under the same
conditions revealed a first gradual denaturation up to 60 °C followed by complete denaturation at
around 60 °C (Figure 3-28b).
70
Kirstin A. Zettlitz
Results
Figure 3-28: Analysis of the melting point of ATROSAB (a) and IZI-06.1 scFv (b) by dynamic light scattering. Dynamic light
scattering intensity (kcps) was measured while temperature was increased in 1 °C intervals.
3.2.5
Affinity measurements
3.2.5.1 IZI-06.1 monovalent antibody constructs
The affinities of IZI-06.1 scFv and IZI-06.1 scFv-HSA for human TNFR1 were determined by quartz
crystal microbalance measurements (QCM, Attana A100 C-Fast system) using immobilized TNFR1-Fc
(Figure 3-29). For the IZI-06.1 scFv an affinity of 2.2 nM was determined (Table 3-4). The scFv-HSA
fusion protein exhibited a 10-fold reduced affinity as compared to the scFv molecule indicating some
sterical hindrance of binding through the HSA fusion partner.
Figure 3-29: Determination of affinities of IZI-06.1 scFv (a) and IZI-06.1 scFv-HSA (b) to human TNFR1-Fc by quartz crystal
microbalance measurements.
Table 3-4: Summary of binding kinetics of IZI-06.1 scFv and scFv-HSA on immobilized huTNFR1-Fc.
construct
IZI-06.1 scFv
IZI-06.1 scFv-HSA
kon (M-1s-1)
3.541 x 10
5
8.284 x 10
4
koff (s-1)
KD (nM)
B (Hz)
res Ssq
-4
2.236
7.212
0.3066
-3
17.4
23.81
0.4460
7.584 x 10
1.443 x 10
71
Kirstin A. Zettlitz
Results
3.2.5.2 ATROSAB, H398
Competition experiments for binding of H398 and ATROSAB to human and rhesus TNFR1-Fc showed
that the two antibodies compete for the same epitope on the respective receptor (Figure 3-30).
These experiments also indicated that, on a molar basis, 5 to 10-fold more ATROSAB was required to
compete binding of H398 to human TNFR1-Fc and 4 to 5-fold more for rhesus TNFR1-Fc. The other
way around only 0.5 – 1.8-fold molar excess of H398 is required to compete binding of ATROSAB to
human TNFR1-Fc (0.2 to 1-fold for rhesus TNFR1-Fc).
Figure 3-30: Competition experiment. Either human TNFR1-Fc (a,b) or rhesus TNFR1-Fc (c,d) was coated (100 ng/well)
and incubated with a constant concentration of either H398 (2 nM; a,c) or ATROSAB (10 nM; b,d). Binding was competed
by increasing concentrations of ATROSAB (a,c) or H398 (b,d). (n=2, shown is experiment 2)
The affinity of ATROSAB for TNFR1 was determined by quartz crystal microbalance measurements
(QCM, Attana A100 C-Fast system) using immobilized TNFR1-Fc. ATROSAB bound with subnanomolar affinity to human and rhesus TNFR1-Fc, similar to the affinity of H398 for human and
rhesus TNFR1-Fc (Table 3-5, Figure 3-31).
72
Kirstin A. Zettlitz
Results
Table 3-5: Summary of binding kinetics of ATROSAB and H398.
construct
antigen
kon (M-1s-1)
ATROSAB
huTNFR1-Fc
3.8 x 10
ATROSAB
rheTNFR1-Fc
6.9 x 10
H398
huTNFR1-Fc
3.1 x 10
H398
rheTNFR1-Fc
2.1 x 10
koff (s-1)
KD (nM)
B (Hz)
res Ssq
5
1.3 x 10
-4
0.35
46.6
0.6449
5
6.7 x 10
-5
0.10
34.9
0.3397
5
7.0 x 10
-5
0.23
45.4
0.4229
5
1.0 x 10
-4
0.49
30.4
0.3738
Figure 3-31: Determination of affinity of H398 and ATROSAB for binding to human and rhesus TNFR1-Fc by quartz crystal
microbalance. a) Binding of ATROSAB to human TNFR1-Fc. b) Binding of ATROSAB to rhesus TNFR1-Fc. c) Binding of H398
to human TNFR1-Fc. d) Binding of H398 to rhesus TNFR1-Fc.
3.2.6
Pharmacokinetics
Pharmacokinetics was determined after a single dose (25 µg) i.v. injection into CD1 mice. Both
antibodies showed biphasic elimination from circulation (Figure 3-32). Serum half-life was
determined to be 10.5 ± 2.8 days for ATROSAB and 8.1 ± 1.5 days for H398 (calculated for the 4
terminal time points).
73
Kirstin A. Zettlitz
Results
Figure 3-32: Pharmacokinetics of ATROSAB and H398. Serum clearance of ATROSAB (a) and H398 (b) after a single i.v.
injection of 25 µg purified IgG into the tail vein of CD1 mice (n=3). Serum concentrations of the antibodies were
determined at different time points by ELISA.
3.2.7
Antagonistic activity of TNFR1-specific antibodies
TNF incubation on Kym-1 cells (expressing approximately 3000 TNFR1, 30000 TNFR2, type I cell)
induced cell death (apoptosis) in approximately 80 - 90 % of cells at concentration above 1 ng/ml
(Figure 3-33a, Figure 3-34a).
The antagonistic effects of IZI-06.1 Fab-HSA and scFv-HSA were analyzed for TNF-mediated
cytotoxicity on Kym-1 cells (Figure 3-33). An IC50 value of 60 nM was measured for scFv-HSA. Fab-HSA
did not reach 50 % inhibition, thus no IC50 value could be determined.
Figure 3-33: Inhibition of TNF-mediated cytotoxicity on Kym-1 cells. a) Killing of Kym-1 cells upon TNF stimulation. b)
Inhibition of TNF-mediated cytotoxicity (1.25 ng/ml TNF) on Kym-1 cells by IZI-06.1 scFv-HSA and IZI-06.1 Fab-HSA. Cells
were analyzed after 6 h by crystal violet staining (n=3, for Fab-HSA n=2). Maximum (20 % viability of control) and half
maximum (60 % viability of control) are depicted in dotted lines.
Furthermore, the antagonistic effects of IZI-06.1 Fab-HSA and scFv-HSA were analyzed for TNFmediated IL-8 release from HT1080 cells. However, no accurate IC50 values could be determined from
these assays, although scFv-HSA showed blocking activity (not shown).
74
Kirstin A. Zettlitz
Results
TNF-induced killing of Kym-1 cells was inhibited in a dose-dependent manner by ATROSAB and H398.
In this assay, a TNF concentration was used resulting in 90 % cell killing (1.25 ng/ml) (Figure 3-34a).
Half-maximal killing, i.e. 55 % viable cells, was observed at about 60 nM for ATROSAB and 5-10 nM
for H398, respectively (Figure 3-34b).
Figure 3-34: Inhibition of TNF-mediated cytotoxicity on Kym-1 cells. a) Killing of Kym-1 cells upon TNF stimulation. b)
Inhibition of TNF-mediated cytotoxicity (1.25 ng/ml TNF) on Kym-1 cells by ATROSAB and H398. Cells were analyzed after
6 h by crystal violet staining (n=3). Maximum (10 % viability of control) and half maximum (55 % viability of control) are
depicted in dotted lines.
The effects of ATROSAB and H398 on TNF-induced secretion of IL-6 from HeLa and IL-8 from HT1080
cells, respectively, were investigated by determining cytokine content of cell culture supernatants by
sandwich ELISA. TNF stimulation (4 nM TNF, 200 ng/ml) for 18 h induced a strong increase in IL-6
secretion from HeLa cells in a dose-dependent manner and reached approximately 700 pg/ml (Figure
3-35a). Similarly, TNF induced secretion of IL-8 from HT1080 cells reached approximately 7000 pg/ml
after incubation with 4 nM TNF for 18 h (Figure 3-35b). To examine potential agonistic activity of the
bivalent antibodies ATROSAB and H398 as well as control IgG (human IgG, Sigma), they were
incubated with HeLa or HT1080 cells in the absence of TNF for 18 h. This resulted only in a marginal
induction of cytokine release, peaking at a concentration around 10 nM. Thus, IL-6 levels increased
from 15 pg/ml of untreated cells to 40 to 60 pg/ml, which is an increase of 3 – 6 % compared to the
values reached after treatment with a comparable TNF concentration (4 nM). Similarly, IL-8 levels
increased from 80 pg/ml endogenous production to approximately 200 pg/ml after incubation with
the antibodies, corresponding to approximately 2 % of the value reached after treatment with a
comparable TNF concentration. The negative control (human IgG) had no effect on cytokine release.
Also a Fab fragment derived from ATROSAB by papain digestion did not induce any detectable
increase of cytokine release (not shown). ATROSAB and H398 inhibited release of IL-6 from HeLa cells
and IL-8 from HT1080 cells induced by 20 pM TNF (1 ng/ml) in a dose-dependent manner. In these
assays, the IC50 values were 60 nM for ATROSAB and 6 nM for H398 for inhibition of IL-6 release
75
Kirstin A. Zettlitz
Results
(Figure 3-35c) and for inhibition of IL-8 release (Figure 3-35d), respectively. In the same assay using
40 pM TNF (2 ng/ml) IC50 values of 760 nM for ATROSAB and 200 nM for H398 for inhibition of IL-6
release (n=2, not shown) and IC50 values of 210 nM for ATROSAB and 62 nM for H398 for inhibition of
IL-8 release (n=3, not shown) were determined.
Figure 3-35: Inhibition of IL-6 and IL-8 secretion by ATROSAB and H398. TNF induced secretion of IL-6 from HeLa cells (a)
and IL-8 from HT1080 (b) in a concentration-dependent manner. Secretion of IL-6 (c) and IL-8 (d) induced by TNF at 1
ng/ml was inhibited by ATROSAB and H398 (n=3).
To further investigate possible agonistic activities of ATROSAB and H398, the antibodies were
incubated in serial dilution with human PBMCs (1 x 105 cells/well) isolated from buffy coat of healthy
donors for 24 h and cytokine concentrations were determined by ELISA as described above. As
control also TNF was incubated with PBMCs for 24 h, in this experiment only a moderate increase of
cytokine levels (IL-6 and IL-8) was observed (Figure 3-36a,c). For IL-6 release a slightly increased level
was determined after incubation with TNF and anti-TNFR2 antibody 80M2 (constant 1 µg/ml),
indicating TNFR2 mediated activation. Neither ATROSAB nor H398 induced interleukin production
(Figure 3-36b,d). These findings indicate that ATROSAB and H398 do not lead to activation of PBMCs,
i.e. are not agonistic in this setting with freshly prepared primary cells.
76
Kirstin A. Zettlitz
Results
Figure 3-36: PBMCs were incubated with TNF or TNF plus cross-linking mAb 80M2 and IL-6 (a) and IL-8 (c) levels were
determined after 24 h by ELISA. In parallel, PBMCs were incubated with ATROSAB, H398 or negative control human IgG
and IL-6 (b) and IL-8 (d) release was determined after 24h (n=3).
Possible agonistic activity was additionally investigated using purified human granulocytes (PMN)
which were freshly prepared from 20 ml blood and purified using Leukoprep (Polymorphprep™, AxisShield, Oslo, Norway) (experiment carried out by Dr. rer. nat. Dafne Müller). PMNs (2 x 10 5 cells)
were incubated with TNF, ATROSAB or human IgG as control at concentrations of 1, 10, and 100 nM
for 1 h. Incubation with phorbolester (PMA) was included as positive control (not shown).
Degranulation was determined by adding dihydro-rhodamine (DHR) and subsequent FACS analysis
gating the granulocyte population (Figure 3-37).
Figure 3-37: Analysis of degranulation from PMNs of a healthy donor. PMNs were incubated with antibodies or TNF for
1 h and degranulation was determined by staining with DHR. Cells were quantified by flow cytometry.
The positive control (PMA) induced nearly complete degranulation (97.7 %, not shown) but TNF
induced as well a significant degranulation reaching approximately 40 % of positively stained cells at
10 – 100 nM. For ATROSAB, no or only a very weak degranulation was observed, compared to the
negative control huIgG.
77
Kirstin A. Zettlitz
3.2.8
Results
Epitope mapping
Because H398 and ATROSAB do not bind to mouse TNFR1, a domain swapping strategy was applied
for epitope mapping (Figure 3-38a). Binding to these chimeric TNFR1-Fc molecules was analyzed by
ELISA. No binding was seen with chimeric molecules where the human CRD1 and 2 (construct 3) or
only the CRD1 was substituted by the corresponding mouse domains (construct 4). Some weak
binding was seen when only the A1 domain of CRD1 of human TNFR1 was exchanged with the
corresponding mouse sequence (construct 5). Binding was also reduced with mouse TNFR1
containing the human CRD1 (construct 7) or the human A1 domain of CRD1 (construct 6), indicating
that further regions are required for full binding. Extension of the human portion to include subdomain A1 of CRD2 resulted in a chimeric TNFR1 to which H398 and ATROSAB show strong binding
(construct 8). Thus, the epitope resides in the N-terminal region of TNFR1 covering residues 1 to 70.
Within this region, 15 residues are different between human TNFR1 and mouse TNFR1, while only
one residue is different between human and rhesus TNFR1 (Figure 3-38b). This residue (Ile 21) is
substituted by a valine in rhesus and mouse TNFR1. Several of the residues different between human
and mouse TNFR1 are exposed in the interaction site of the receptor with TNF, including Pro23,
Gln24, Tyr30, Asn31, Ser57, Ser 59, His66, and His69. In order to further narrow down the epitope,
residues P23 and Gln24, located in sub-domain A1 of CRD1, were exchanged by the corresponding
mouse residues in the chimeric TNFR1 h1-2A1/m2B2-4 (construct 10, Figure 3-38a,b). These
mutations completely abolished binding of ATROSAB and strongly reduced binding of H398 under the
applied assay conditions.
Figure 3-38: a) Epitope mapping of ATROSAB and H398 using wild-type and chimeric human/mouse TNFR1-Fc fusion
proteins. b) Sequence comparison of the identified epitope region (aa 1-70) of human (huTNFR1), mouse (moTNFR1), and
rhesus (rhTNFR1) TNFR1. Cysteine residues are marked with grey boxes and the 2 positions (P23, Q24) analyzed by sitedirected mutagenesis are marked by asterisks.
78
Kirstin A. Zettlitz
Results
The structure of TNFR1 shows the TNF contact site which is mainly located at CRD2 and 3 and shows
that the antibody binding site at least partially overlaps with TNF binding (Figure 3-39). Thus,
displacement of TNF by ATROSAB could be explained by sterical hindrance.
Figure 3-39: a) Structure of TNF (red) bound to TNFR1 (blue). The identified epitope region is marked in green. b) A single
TNFR1 chain. The positions (P23, Q24) identified by site-directed mutagenesis to contribute to binding of ATROSAB and
H398 are highlighted in dark green. Structures were visualized with PyMol (http://www.pymol.org).
3.2.9
Optimization of IZI-06.1
Antibody affinity plays an important role in biological efficacy and increased affinity may allow for a
reduced dosage of a therapeutic antibody and thereby toxic side effects may be reduced. The
molecular surface of the antibody-combining site of an antibody is formed by the intersection of the
apical regions of VL and VH (Figure 3-40a). The CDRs (complementarity-determining regions) lie in
close spatial proximity and provide a contiguous surface for antigen recognition. The VH-CDRs, and
the VH-CDR3 in particular, generally make more extensive contact than the VL-CDRs, and the
geometrical centre of the antibody-antigen interface tends to lie near VH-CDR3. That is the initially
reason for excluding VH-CDR3 and VL-CDR3 from optimization. The combining site of antibodies that
recognize large protein antigens are generally more planar than those of antibodies recognizing
smaller antigens such as peptides or DNA which have a grooved antigen-contacting surface. Large
antigens often contact antibody residues at the edge of the combining site and interact with more
apical portions of the CDR-loops. There exists a strong correlation between residues that do not form
contacts with antigen and those residues that are important in defining the canonical backbone
structures of the CDR loops. These residues tend to pack internally and are therefore less exposed on
the antibody-combining site surface. Taken these considerations together exposed residues of VHCDR1,2 and VL-CDR1,2 were chosen for site directed mutagenesis (Figure 3-40b).
79
Kirstin A. Zettlitz
a)
Results
b)
Figure 3-40: Antigen binding site of IZI-06.1 scFv (view is looking down). a) The CDRs of the heavy chain are depicted in
red, the CRDs of the light chain are depicted in blue. b) Exposed residues of CDRs 1 and 2 which were chosen for
mutagenesis. Structures were visualized with PyMol (http://www.pymol.org).
Because selection of affinity matured mutants of IZI-06.1 was to be done by phage display, correct
display of scFv fragments on phage was confirmed by performing a phage rescue of IZI-06.1_wt scFv
(wild type) and showing specific binding of phage to human TNFR1-Fc in ELISA (Figure 3-41).
Figure 3-41: Binding of IZI-06.1 scFv-phage to immobilized huTNFR1-Fc in ELISA. Immobilized HSA was included as
control. Bound antibody –phage were detected by HRP-conjugated anti-M13 antibody.
3.2.9.1 Library construction
Mutagenized scFv libraries were generated by site directed mutagenesis using primer introducing
mutations and restriction sites for cloning into phage display vector pHEN2_scFvIZI-06.1_notwt (this
acceptor vector was modified to contain a deletion after CDRH2 and an additional stop-codon to
prevent production of wild type scFv). The primer contain the degenerate codon NHK (N=G,C,A,T /
H=A,T,C / K=G,T) at the positions to be randomized, this allows for 24 codons = 16 aa (possible amino
acids: LIMV FY KH DE STQN AP; absent amino acids: CWRG). Four different libraries were generated
by using combinations of the primers. The PCR-based mutagenesis and cloning strategy is depicted in
Figure 3-42.
80
Kirstin A. Zettlitz
a)
Results
b)
Figure 3-42: Cloning strategy. a) Primer introducing mutations and restriction sites for subsequent cloning. b) Library
construction. Combination of primer resulted in four libraries. Calculated permutations are depicted in red.
Size of final libraries (Table 3-6) was determined by plating serial dilutions of transformed E. coli TG1.
Diversity of the libraries (frequency and distribution of mutations) was confirmed by sequencing of
randomly chosen clones (shown as percent functionality in Table 3-6).
Table 3-6: Size and functionality of generated libraries.
library 1
library 2
library 2a
library 2b
mutations in
CDRH1/CDRL2
CDRH2/CDRL1
CDRH2
CDRL1
positions
6
12
7
5
H31,32,33/L50,53,56
H50,52,53,54,56,58,61
H50,52,53,54,56,58,61
L27D,27E,28,30,32
L27D,27E,28,30,32
possible
diversity
7
2.8 x 10
7
1.6 x 10
14
2.68 x 10
8
1.05 x 10
6
8
8
7
size
6.4 x 10
2.2 x 10
2.57 x 10
7.95 x 10
functionality
90 %
67.5 %
73 %
60 %
3.2.9.2 Immunotube selection of library 1 (CDRH1/CDRL2)
IZI-06.1 scFv library 1 (L1) was selected on huTNFR1-Fc and moTNFR1-Fc coated immunotubes using
conditions of high stringency (i.e. low amounts of coated antigen, long washing steps). Binding of
enriched phage antibodies to huTNFR1-Fc and moTNFR1-Fc was confirmed by polyclonal phage
ELISA. No signals were found for phage selected on moTNFR1-Fc indicating that mutations at these
positions are not able to confer binding to mouse TNFR1.
Clones from round 2 and 3 were screened for binding to huTNFR1-Fc and moTNFR1-Fc using soluble
scFv produced in non-suppressor strain HB2151. In total 27 positive clones (19 different in sequence,
see Table 3-7) were identified out of 192 screened.
81
Kirstin A. Zettlitz
Results
Table 3-7: Sequences of individual clones selected from library 1 (immunotube selection).
CDRH1
D
F
Y
position
L1 hu_A1
L1 hu_G3
L1(2) hu_A1
L1(2) hu_G3
L1 hu_A4
L1(2) hu_F5
L1 hu_D3
L1 hu_B5
L1 hu_E2
L1 hu_F10
L1 hu_E3
L1 hu_C5
L1 hu_A11
L1(2) hu_G6
L1 hu_E4
L1 hu_D1
L1 hu_H4
L1 hu_H5
L1 hu_E12
H31
T
T
T
T
D
L
S
E
S
D
E
Q (tag)
E
D
S
Y
Q
A
A
H32
Y
Y
F
F
Y
Y
Y
D
F
F
F
F
N
N
N
M
M
A
H
H33
F
F
F
F
F
L
L
L
Y
Y
V
F
M
M
M
Y
M
F
L
IN
CDRL2
T
L1 hu_A1
L1 hu_F10
L1(2) hu_F5
L1(2) hu_G3
L1(2) hu_A1
L1(2) hu_G6
L1 hu_E12
L1 hu_E2
L1 hu_B5
L1 hu_C5
L1 hu_E4
L1 hu_H4
L1 hu_H5
L1 hu_E3
L1 hu_A4
L1 hu_G3
L1 hu_D3
L1 hu_A11
L1 hu_D1
L50
N
N
N
N
N
N
N
N
N
Q
Q
Q
Q
Q
T
T
T
E
E
VS N
RF S
L53
K
K
K
K
K
A
A
N
Q
K
K
K
K
Q
H
Q
K
K
Q
L56
T
T
T
T
M
E
A
K
K
N
K
M
Q
A
H
H
S
N
K
Sequence analysis of enriched amino acids at the six randomized positions in CDRH1 and CDRL2 of
these clones (Figure 3-43) revealed a preference for certain residues at some positions, e.g. Asn or
Gln at position L50 and Lys at position L53. Furthermore, in CDRH1 a preference for aromatic
residues was observed at position H32 and H33, i.e. retaining the original pattern, while at position
H31 a trend towards hydrophilic residues (T, D, S, E) was observed.
Figure 3-43: Sequence analysis of the selected clones of library 1 (CDRH1/CDRL2). Changed amino acids in CDRH1 are
shown in red, changed amino acids in CDRL2 are shown in blue. Recurrence of original amino acids (present in
IZI-06.1_wt) is indicated in green.
82
Kirstin A. Zettlitz
Results
3.2.9.3 Immunotube selection of library 2 (CDRH2/CDRL1)
IZI-06.1 scFv library 2 (L2) was selected on huTNFR1-Fc and moTNFR1-Fc coated immunotubes as
described for L1. However, screening of individual clones of round two and three did not result in the
identification of positive clones. Presumably, randomized positions affected one or more critical
residues, which were not present in the correct composition in library 2 covering only a small fraction
of all possible combinations. Therefore, library 2 was subdivided into two libraries L2a (CDRH2) and
L2b (CDRL1).
3.2.9.4 Immunotube selection of library 2a (CDRH2)
IZI-06.1 scFv library 2a (L2a) was selected on huTNFR1-Fc and moTNFR1-Fc coated immunotubes as
described for libraries L1 and L2. In total 65 clones positive for huTNFR1-Fc binding were observed
out of 282 screened, thereof 24 clones were sequenced and 21 positive clones (16 different in
sequence, see Table 3-8) were identified. No clones were found that bind to moTNFR1-Fc. An analysis
of the frequency of enriched residues revealed a preference for certain residues at some positions
(Figure 3-45). At position H50 and H61 the original residue reoccurred, i.e. Glu at H50 and Glu (or the
conserved Asp) at H61. New residues were selected for the remaining 5 residues, with for example a
strong preference for Lys at position H52 (Tyr in wt) and Lys at position H58 (Tyr in wt). None of the
wild type residues were enriched at these 5 positions.
Table 3-8: Sequences of individual clones selected from library L2a (immunotube selection).
CDRH2
WIG E
I Y
P Y
S
G H
A Y
Y N E
position
Lib2a_hu2R_C2
Lib2a_hu2R_C4
Lib2a_hu2R_D8
Lib2a_hu2R_F9
Lib2a_hu2R_C12
Lib2a_hu2R_E2
Lib2a_hu2R_G4
Lib2a_hu2R_F8
Lib2a_hu2R_E6
H50
E
E
K
E
E
E
E
E
E
H52
L
K
V
V
K
K
K
K
K
H53
T
Q
D
T
D
T
I
D
E
H54
Q
E
Q
Q
E
E
E
E
E
H56
T
Q
N
E
T
Q
A
N
T
H58
H
V
K
N
E
Q
H
A
K
H61
E
E
D
E
D
T
K
E
E
Lib2a_hu3R_D12
Lib2a_hu3R_A5
Lib2a_hu3R_D4
Lib2a_hu3R_D11
Lib2a_hu3R_G11
Lib2a_hu3R_B3
Lib2a_hu3R_B2
Lib2a_hu3R_F1
Lib2a_hu3R_H10
E
E
E
E
E
E
E
E
E
K
K
K
E
E
K
K
L
L
E
E
E
H
H
D
D
E
E
E
E
E
T
T
M
M
Q
Q
T
T
T
K
K
A
A
V
T
K
K
K
E
E
K
K
K
N
E
E
E
D
D
A
A
D
P
83
KFK
Kirstin A. Zettlitz
Results
3.2.9.5 Equilibrium selection of library 2a (CDRH2)
IZI-06.1 scFv library 2a (L2a) was also selected using biotinylated antigen in solution. Prior to the first
selection round a negative selection with moTNFR1-Fc was performed. Subsequently, 6 rounds of
selection were performed with decreasing antigen concentrations, phage numbers and Dynabeads
and increasing incubation times and washing steps. Binding of enriched phage antibodies to
huTNFR1-Fc was confirmed by polyclonal phage ELISA (Figure 3-44).
Figure 3-44: Polyclonal phage ELISA of selection rounds of L2a on huTNFR1-Fc (antigen concentrations indicated) with
increased incubation times and washing steps.
From 2nd – 4th round 186 clones were screened, out of those 38 were found to be positive for binding
to huTNFR1-Fc. 20 clones were sequenced and 9 unique clones were identified (Table 3-9). From the
5th and 6th round 27 (R5) and 67 (R6) positive clones were identified out of 141 screened from each
round. 20 clones were sequenced and were found to be identical in CDRH2-sequence (Table 3-9).
Table 3-9: Sequences of individual clones selected from library 2a (equilibrium selection).
CDRH2
WIG E
I Y
P Y
S
G H
A Y
Y N E
position
Lib2a_huB2R_B1
Lib2a_huB2R_E6
Lib2a_huB2R_F1
Lib2a_huB2R_F4
H50
E
E
E
E
H52
E
Q
K
K
H53
E
A
A
N
H54
Y
H
E
D
H56
P
T
E
E
H58
K
T
D
E
H61
D
E
D
K
Lib2a_huB3R_A9
Lib2a_huB3R_H10
E
E
Q
H
K
P
A
K
P
H
K
T
Q
E
Lib2a_huB4R_B6
Lib2a_huB4R_B9
Lib2a_huB4R_G6
E
E
E
K
K
K
D
T
E
E
E
E
T
N
T
Q (tag)
T
K
K
A
E
Lib2a_huB5R_IA2
Lib2a_huB5R_IID3
E
E
V
V
T
T
Q
Q
E
E
K
K
D
D
Lib2a_huB6R_IE2
Lib2a_huB6R_IA11
Lib2a_huB6R_IG11
Lib2a_huB6R_IH6
Lib2a_huB6R_IIA10
Lib2a_huB6R_IIG12
E
E
E
E
E
E
K
V
V
V
V
V
D
T
T
T
T
T
E
Q
Q
Q
Q
Q
T
E
E
E
E
E
Q (tag)
K
K
K
K
K
K
D
D
D
D
D
84
KFK
Kirstin A. Zettlitz
Results
The analysis of the frequency of enriched amino acid residues resulted in a similar pattern as for the
immunotube selection (Figure 3-45). At position H50 and H61 the original residue reoccurred, i.e. Glu
at H50 and Glu (or the conserved Asp) at H61. New residues were selected for the remaining 5
residues. A difference between the two selection methods was seen at position H52 (Tyr in wt), at
which Val was preferred (Lys in immunotube selection). The strong preference for Lys at position H58
(Tyr in wt) was seen in both selections. None of the wild type residues were enriched at these 5
positions.
Figure 3-45: Sequence analysis of the selected clones of library 2a (CDRH2). Changed amino acids in CDRH2 are shown in
red. Recurrence of original amino acids (present in IZI-06.1_wt) is indicated in green.
3.2.9.6 Immunotube selection of library 2b (CDRL1)
IZI-06.1 scFv library 2b (L2b) was selected on huTNFR1-Fc and moTNFR1-Fc coated immunotubes
using conditions of high stringency as described for L1 and L2a. Binding of enriched phage antibodies
to huTNFR1-Fc was confirmed by polyclonal phage ELISA. Clones from round 1 and 2 were screened
for binding to huTNFR1-Fc and moTNFR1-Fc using soluble scFv produced in non-suppressor strain
HB2151. In total 67 positive clones were observed out of 271 screened, thereof 48 clones were
sequenced and 21 positive clones (17 different in sequence) were identified. Amino acids at the 5
85
Kirstin A. Zettlitz
Results
randomized positions in CDRL1 of these clones are shown in Table 3-10. No clones were found that
bind to moTNFR1-Fc.
Table 3-10: Sequences of individual clones selected from library 2b (immunotube selection).
CDRL1
RSSQSLL H
position
Lib2b_hu1R_F3
Lib2b_hu1R_E2
Lib2b_hu1R_F7
Lib2b_hu1R_D10
Lib2b_hu1R_D11
Lib2b_mo2R_F7
Lib2b_mo2R_D9
Lib2b_hu1R_E6
Lib2b_hu1R_D8
Lib2b_hu1R_A11
Lib2b_hu1R_G5
Lib2b_hu1R_E11
Lib2b_hu2R_IH1
Lib2b_hu2R_E3
Lib2b_hu2R_C12
Lib2b_hu1R_F10
Lib2b_hu1R_C10
Lib2b_hu1R_E7
Lib2b_hu1R_H9
Lib2b_hu2R_B4
Lib2b_hu1R_C8
L27D
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
T
L
L
D
N
S
N
L27E
D
D
D
D
D
V
V
S
S
S
A
A
T
T
M
P
P
P
S
T
T
L28
S
S
S
S
S
N
N
S
S
H
T
H
N
S
D
A
S
S
N
H
A
G N
L30
L
L
L
L
L
V
V
Q
I
A
L
L
I
V
S
A
L
V
S
L
V
T Y
LH
L32
F
F
F
F
F
P
P
L
L
A
Y
S
Y
K
M
Y
P
P
P
Q
L
An analysis of the frequency of enriched residues of clones selected from L2b revealed a preference
for certain residues at some positions (Figure 3-46). Thus, a strong preference for Ser was found at
position L27D (His in wt) and position L28 (Asn in wt). A preference for hydrophobic residues was
seen at position L30. At position L27E, L28 and L32 the original amino acid residues (as present in the
wild type) reappeared to a small extent.
86
Kirstin A. Zettlitz
Results
Figure 3-46: Sequence analysis of the selected clones of library 2b (CDRL1). Changed amino acids in CDRL1 are shown in
blue. Recurrence of original amino acids (present in IZI-06.1_wt) is indicated in green.
3.2.9.7 Summary of selections of CDR H1, H2, L1, L2
A summary of the results from selections of library 1, 2a and 2b on huTNFR1-Fc is shown in Figure
3-47.
Figure 3-47: Summary of enriched residues in CDRL1, L2, H1, H2. Original and the selected prevalent residues are shown.
Structures were visualized with PyMol (http://www.pymol.org).
87
Kirstin A. Zettlitz
Results
3.2.9.8 ScFv IZI-06.1_mutants and combined scFv
Individual clones as well as scFv molecules combining all preferred residues (scFv kze1-4) were
subcloned into expression vector pAB1, produced in E. coli TG1 and purified by IMAC. Table 3-11
shows the amino acid sequence of the CDRs of the different scFvs.
Table 3-11: Differences in CDRs of scFvIZI-06.1 mutants and combined scFvs and production yields. The origin of the CDRs
in the combined scFvs are indicated in parentheses (H1/H2/L1/L2).
name
CDRH1
CDRH2
CDRL1
CDRL2
scFv IZI-06.1_wt
DFYIN
WIGEIYPYSGHAYYNEKFK
SLLHSNGNTYLH
LLIYTVSNRFS
yield
[mg/l]
0.32-0.7
scFvL1(2)_hu2R_G3
TFFIN
WIGEIYPYSGHAYYNEKFK
SLLHSNGNTYLH
LLIYNVSKRFT
0.36
scFvL2a_huB6R_IG11
DFYIN
WIGEIVPTQGEAKYNDKFK
SLLHSNGNTYLH
LLIYTVSNRFS
0.94 ± 0.3
scFvL2a_hu3R_A5
DFYIN
WIGEIKPEEGTAKYNEKFK
SLLHSNGNTYLH
LLIYTVSNRFS
0.87
scFvL2b_hu1R_F3
DFYIN
WIGEIYPYSGHAYYNEKFK
SLLSDSGLTFLH
LLIYTVSNRFS
0.78
scFv kze1
(G3/A5/F3/G3)
scFv kze2
(A1/A5/F3/G3)
scFv kze3
(G3/IG11/F3/G3)
scFv kze4
(G3/IG11/wt/G3)
TFFIN
WIGEIKPEEGTAKYNEKFK
SLLSDSGLTFLH
LLIYNVSKRFT
0.38-0.41
NFMIN
WIGEIKPEEGTAKYNEKFK
SLLSDSGLTFLH
LLIYNVSKRFT
0.58-0.8
TFFIN
WIGEIVPTQGEAKYNDKFK
SLLSDSGLTFLH
LLIYNVSKRFT
0.34
TFFIN
WIGEIVPTQGEAKYNDKFK
SLLHSNGNTYLH
LLIYNVSKRFT
0.5-0.8
3.2.9.9 Binding and activity of scFv IZI-06.1 derivates
Purified scFvs (Figure 3-48a,b; Figure 3-49a) were tested in ELISA for binding to huTNFR1-Fc (Figure
3-48c). ScFv kze3 as well as the four scFv with individually changed CDRs (see Table 3-11) showed
binding to huTNFR1-Fc, while scFv kze1 and kze2 were inactive (not shown). Except for scFv L2b_F3
and scFv L2a_A5, all scFvs showed similar binding as wild type scFv IZI-06.1.
Figure 3-48: Characterization of purified scFvs. a) SDS-PAGE analysis of purified scFvs. 15% SDS-PAGE, reducing
conditions, 2 µg purified scFv/lane, Coomassie staining. b) Western blot analysis of purified scFvs. 15% SDS-PAGE,
reducing conditions, 0.5 µg purified scFv/lane, WB: anti-His-HRP 1/1000, ECL. c) Binding of scFv derivates to human
TNFR1-Fc (1 µg/ml) in ELISA (n=3). Data were normalized to Y=0 becomes 0 % for all data set and the largest value in each
data set becomes 100 %. Data were fitted to the approximate equation of one site – specific binding Y = Bmax * X/(EC50 +
X), where Bmax is the extrapolated maximum specific binding and EC50 is the concentration needed to achieve halfmaximum binding at equilibrium.
88
Kirstin A. Zettlitz
Results
Because CDRL1 seems to have a negative effect on binding, a new scFv kze4 was generated based on
scFv kze3, in which the CDRL1 was converted back to wild type. In ELISA binding to huTNFR1-Fc was
slightly weaker compared to scFv IZI-06.1_wt and scFv kze3.
Figure 3-49: Characterization of scFvkze4. a) SDS-PAGE (15%) analysis of purified scFvkze4 under reducing (1) and nonreducing (2) conditions (Coomassie staining). b) Binding of scFvkze 4 to immobilized huTNFR1-Fc in ELISA. Bound scFv
was detected by HRP-conjugated anti-His antibody (1/2000) (n=3). Data were normalized to Y=0 becomes 0 % for all data
set and the largest value in each data set becomes 100 %. Data were fitted to the approximate equation of one site –
specific binding Y = Bmax * X/(EC50 + X), where Bmax is the extrapolated maximum specific binding and EC50 is the
concentration needed to achieve half-maximum binding at equilibrium.
Affinities of scFv IZI-06.1 derivates were determined by QCM measurements on immobilized
huTNFR1-Fc (Table 3-12). The wild type scFv IZI-06.1 showed an affinity of 2.2 nM. Of the selected
mutants only scFv L2a_IG11 showed an 2-fold higher affinity (1.2 nM) while scFv L1(2)_G3 showed a
comparable affinity of 2.8 nM. Clones scFv L2a_A5 and scFv L2b_F3 showed decreased affinities of
15.2 nM and 44.6 nM, respectively. ScFv kze1 and kze2 (containing CDRH2 of clone L2a_A5 and
CDRL1 of L2b_F3) showed no binding to huTNFR1-Fc in these experiments. ScFv kze3 that contains
CDRH2 of clone L2a_IG11 bound to huTNFR1-Fc with an affinity of 9.7 nM. An affinity of 7.3 nM was
determined for scFv kze4 in which CDRL1 was back-mutated to wild type.
Table 3-12: Summary of binding kinetics of monovalent scFv IZI-06.1 derivates.
sample
n
kon (M-1s-1)
scFvIZI-06.1_wt
5
3.541 x 10
5
1.772 x 10
5
9.484 x 10
scFvkze4
1
koff (s-1)
KD (nM)
B (Hz)
residual Ssq
-4
2.236
7.212
0.3066
-3
7.291
7.839
0.1308
-4
9.687
7.459
0.1626
-4
1.158
8.423
0.2593
-4
2.757
7.809
0.2016
-3
15.17
6.947
0.2370
-3
44.58
7.869
0.1731
7.584 x 10
1.292 x 10
scFvkze3
1
0.979 x 10
5
scFvL2a_IG11
2
2.269 x 10
5
2.606 x 10
scFvL1(2)_G3
1
2.134 x 10
5
5.884 x 10
scFvL2a_A5
1
1.261 x 10
5
1.913 x 10
scFvL2b_F3
2
1.016 x 10
5
3.077 x 10
The antagonistic effects of scFv IZI-06.1_wt, individual clones and designed scFvs were analyzed for
TNF-mediated cytotoxicity on Kym-1 cells (Figure 3-50). Of the individual clones only scFv L2a_IG11
89
Kirstin A. Zettlitz
Results
showed a higher blocking activity (IC50 value: approximately 10 nM) compared to scFv IZI-06.1_wt
(IC50 value: approximately 30 nM). ScFv L1(2)_G3 showed similar blocking activity (IC50 value:
approximately 30 nM) compared to scFv IZI-06.1_wt. Both scFv L2b_F3 and scFv L2a_A5 showed an
about 300-fold reduced blocking activity (IC50 value: approximately 1000 nM) and scFv kze3 (which
comprises CDRL1 of L2b_F3) showed no blocking activity at all, although ELISA and QCM experiments
showed binding to TNFR1-Fc. The modified variant scFv kze4 (CDRL1 mutated back to the wild type
residues) on the other hand, showed blocking activity although to a weaker extend compared to the
wild type scFv IZI-06.1 (IC50 value: approximately 200 nM).
Figure 3-50: Inhibition of TNF-mediated cytotoxicity on Kym-1 cells. a) Killing of Kym-1 cells upon TNF stimulation. b) IC50
values of scFvs. c,d) Inhibition of TNF-mediated cytotoxicity (1.25 ng/ml TNF) on Kym-1 cells by IZI-06.1 scFv mutants.
Cells were analyzed after 7 h by crystal violet staining (n=3). Maximum (40 % viability of control) and half maximum (70
% viability of control) are depicted in dotted lines.
90
Kirstin A. Zettlitz
Discussion
4 Discussion
4.1 Hsp70-specific antibodies for cancer therapy
4.1.1
Chimerization of cmHsp70.1 (IgG tumex)
In this study, the variable regions of mouse antibody cmHsp70.1 were cloned and various
recombinant antibodies (scFv, scFv-Fc, chimeric IgG) could be produced in E. coli or mammalian cells.
For all of them, an identical antigen binding activity was demonstrated as compared to the parental
antibody cmHsp70.1. A sequence analysis revealed no differences between the variable lambda light
chain domain and the closest mouse Vλ germline gene. In contrast, 8 differences were found
between the cloned VH sequence and the closest mouse V germline gene, 7 of them located in
framework regions (FR1, FR3) and only one in CDR1. The first three substitutions at positions 1, 3,
and 5 are most likely due to cloning artifacts caused by the degenerate primer used for amplification
of the VH sequence. Because production and/or antigen binding might be influenced by these
alterations, a variant (variant 1) having these 3 residues exchanged by the germline residues was
produced. However, both the originally cloned sequence as well as the variant (variant 1), showed an
identical binding behavior in ELISA when produced as scFv molecules in E. coli, indicating that these
residues have no influence on the antigen binding activity (Seitter 2010). Also, no substantial
improvement of expression in E. coli for variant 1 was observed.
A possibility to improve the expression of scFv tumex might be the co-expression of chaperones, as
already shown for various other scFv molecules (Bothmann and Pluckthun 2000; Ow et al. 2010).
Recently, Friedrich and co-workers described cloning of the cmHsp70.1 variable domain (Friedrich et
al. 2010) and they also observed low production levels in E. coli for recombinant chimeric Fab
fragments including sequence-optimized derivatives, e.g. generated by correcting, as in this study,
the N-terminal sequence of the cloned VH and VL domains to that obtained by Edman sequencing and
by codon optimization of two rare codons (encoding V47 and V48) of the VL domain. In their study,
yields were dramatically increased (more than 100-fold) by co-expression of two thiol-disulfide
oxidoreductases (DsbA, DsbC) and two peptidyl-prolyl cis/trans-isomerases with chaperone activity
(FkpA, SurA) indicating that disulfide bond formation and cis/trans-isomeriation of prolyliminopeptide bonds are the rate-limiting steps (Friedrich et al. 2010).
In initial experiments, low production yields were also found for the scFv-Fc and IgG molecules
composed of the variable mouse sequences. An extensive screening of stably transfected cells was
required to identify clones with an improved production of tumex IgG. In the case of scFv-Fc and
91
Kirstin A. Zettlitz
Discussion
chimeric IgG produced by mammalian cells through the secretion process, the protein should be
correctly folded and include the internal disulfide bond formation also in the variable domains. These
results indicate that the low production yields are not solely due to disulfide bond formation, but
also other disadvantages may result from the mouse variable domains.
Both the scFv-Fc tumex and the chimeric IgG tumex recognized recombinant human Hsp70 (rhHSP70SBD) in ELISA with similar sensitivity as cmHsp70.1 and showed crossreactivity with mouse Hsp70 in
immunoblot experiments, thereby confirming that the antigen-binding site of cmHsp70.1 was
successfully cloned.
4.1.2
Humanization of cmHsp70.1 (IgG humex)
In this study the variable region of the Hsp70 specific antibody cmHsp70.1 were successfully
humanized through CDR grafting. In this strategy, one usually chooses a framework sequence that is
most similar to the murine donor sequence in order to avoid differences in core packing and
framework confirmation, which could have a disadvantageous effect on CDR conformation and thus
on antigen affinity (Worn et al. 2000). A sequence identity of at least 65 % has been suggested for a
successful humanization process (Hinton et al. 2004), as the antigen–binding affinity of the
humanized antibody correlates with the degree of homology between the human and murine
framework sequences.
Beside the overall homology, key positions play a crucial role for successful humanization, e.g.
residues at the VH/VL interface or in the Vernier zone can have a significant impact on antigen
binding. This is especially true for murine Vλ domains, which show several amino acid substitutions at
the VH/VL interface compared with the closest human homologue (Ewert et al. 2004). In this work,
the subclass of the murine λ light chain donor framework was retained and a human germline λ
sequence with identical canonical structure and a similarity of 67.27 % (V3-2, IGLV7-4*01) was
chosen. At two positions, which are important for VH/VL interface, the donor and acceptor framework
sequences differed: position L44 is a Phe in VL tumex and a Pro in V3-2, position L87 is a Phe in VL
tumex and a Tyr in V3-2. Because position L44 is one of six residues that constitute the core of the
VH/VL interface (Al-Lazikani et al. 1997) and Pro is the by far most frequently occurring amino acid at
this position, the mouse residue was not retained. At position L87 both Phe and Tyr occur, but Tyr
with more than two-fold higher frequency compared to Phe, which was the reason to keep the
residue (Tyr) present in the human acceptor germline sequence. So far, there are very rare examples
of successful antibody humanization with a human λ chain serving as acceptor (Schlapschy et al.
2009). A reason for that may be that κ chains are more frequent in mouse antibodies than λ chains
(Mole et al. 1994).
92
Kirstin A. Zettlitz
Discussion
Similarity search for the VH tumex revealed a number of human germline genes with high homology.
The germline sequence showing the highest homology (IGHV2-70: 67.12 % similarity, z-score: -1.793)
and the germline sequence showing the highest humanness (IGHV4-28: 66.44 % similarity, z-score:
0.143) were chosen for virtual CDR grafting and modeling. Probably because both acceptor
sequences were of distinct canonical classes, they failed to retain the canonical structure in the
modeled humanized version. Therefore, an additional similarity search was performed for each FR
separately and a human germline sequence was chosen which was less homologue (IGHV3-66, DP86: FR1: 53.3 %, FR2: 84.6 %, FR3: 56.2%) but of the identical canonical class as VH tumex.
The Vernier zone residues are located in the β-sheet FR regions closely underlying the CDRs and have
been known to critically affect antigen binding by directly contacting the antigen, affecting CDR loop
conformation, and/or influencing packing interactions between β-sheet strands (Foote and Winter
1992; Al-Lazikani et al. 1997; Makabe et al. 2008). The donor Vernier zone residues probably directly
contacting the antigen (covered by the contact CDR definition) were preserved at sites where the
sequences differed, while the Vernier zone residues located in the FR regions (FR3 of each of the
heavy and the light chain) were changed to those present in the acceptor framework sequence.
Humanization of cmHsp70.1 resulted in variable domains with improved production in E. coli and
thermal stability, which also translated into improved production of whole IgG in mammalian cells,
with initial cell pools of stable transfected cells already producing more than 23 mg/l supernatant
and no need for extensive screening. Although it cannot be excluded that integration position effects
may contribute to net expression in mammalian cells. Nevertheless, this finding confirms that
humanization can have a beneficial effect on productivity and stability (Damschroder et al. 2007;
Honegger 2008) and that rather framework residues than the CDRs account for the poor production
of the mouse variable domains.
Importantly, specificity and affinity was not affected by CDR grafting onto closely related human
germline sequences. No back-mutations of mouse framework residues were necessary, while in
literature it is described that the generation of humanized antibodies often results in a decrease of
affinity (Saldanha et al. 1999; Schlapschy et al. 2004) and generally requires additional transfer of one
or more framework-region residues from the parent mouse antibody (Gonzales et al. 2005). Other
humanization methods have been described, like humanization by resurfacing first described by
Padlan (Padlan 1991). The murine framework surface residues that are not observed in human
antibodies are changed based on the observation that the immunogenicity of a protein is determined
by the accessible and protruding residues (Novotny et al. 1986). However, CDR grafting remains the
93
Kirstin A. Zettlitz
Discussion
most successful applied method and the approved humanized therapeutic antibodies were all
generated by CDR grafting.
Interestingly, the VH sequence of cmHsp70.1 contains an N-glycosylation site (N – X - S) at position 68
of FR3 of the VH domain, not found in the closest mouse germline sequence. It is established that
20 – 30 % of polyclonal human IgG molecules bear N-linked oligosaccharides within the Fab region
(Jefferis 2005). Such variable domain N-glycans can influence antigen binding and also neutralizing
activity of antibodies (Wright and Morrison 1993; Jacquemin et al. 2006). For example, it was shown
that removal of a N-glycosylation site at position 73 of the VH domain during humanization of antiCD33 monoclonal antibody M195 led to a several fold increase in affinity (Co et al. 1992; Co et al.
1993). In other cases, e.g. humanization of anti-p185Her2 monoclonal antibody 4D5 containing a Nglycosylation site at position 65 of the VL domain, no differences in affinity were found between the
glycosylated and aglycosylated form of this antibody (Carter et al. 1992). Glycosylation at position 68
of IgG tumex VH domain was indicated by an increased reduction of the molecular mass (4 kDa) of
the heavy chain of IgG tumex after PNGase treatment as compared to IgG humex, which lacks this Nsequon and showed only a reduction by 2 kDa, caused by removal of the N-glycan in the CH2 domain.
Binding to Hsp70 in ELISA was observed for the bacterially produced scFv tumex (i.e. not
glycosylated) as well as scFv-Fc tumex produced in HEK293 cells. Furthermore, affinity of humex IgG
was identical to that of cmHps70.1 and tumex IgG, indicating that the VH N-glycan does not
contribute to antigen binding.
Beside influencing antigen binding, glycosylation can also cause anaphylactic reactions as reported
for cetuximab (Chung et al. 2008). Hypersensitivity to cetuximab, is caused by preexisting IgE
antibodies against an oligosaccharide present on the recombinant molecule at Asn88 in the Fab
portion (heavy chain) with a range of sugars, including galactose-α-1,3-galactose. Cetuximab is
produced in the mouse cell line SP2/0 (unlike most other mAb), which expresses the gene for α-1,3galactosyltransferase (Koike et al. 2007). In conclusion, IgG-Fab glycosylation can impact differentially
on the structural and functional characteristics of IgG and for a therapeutic antibody it might be
advantageous if controlling of glycoform fidelity is restricted to one site (in the IgG-Fc region).
4.1.3
Epitope mapping of Hsp70-specific antibodies
The mouse monoclonal antibody cmHsp70.1 (clone C92F3B1; (Gehrmann et al. 2004)) was raised by
repeated immunization with the Hsp70 peptide TKDNNLLGRFELSG (TKD peptide; aa 450-463) with
complete Freud's adjuvants and screening for reactivity with the inducible 72 kDa heat shock protein
(HSPA1A, human gene 3303) but not with Hsc70 protein (HSPA8, human gene 3312) in Western blot
analysis, and for its capacity to react with cell surface-bound Hsp70 on viable tumor cell lines
94
Kirstin A. Zettlitz
Discussion
(Multhoff 2007). By peptide scanning (pepscan analysis), the sequence NLLGRFEL within the TKD
peptide was identified as the relevant portion of the cmHsp70.1 epitope (Multhoff 2007). Contrary to
that, this study showed that soluble peptides comprising this TKD sequence were not capable of
inhibiting binding to Hsp70 in ELISA of cmHsp70.1 as well as the chimeric and humanized derivative.
Also, recombinant fragments of Hsp70 containing this peptide sequence but terminating at residue
475 and 495, respectively, (fragments 2 and 8, Figure 3-16) did not show binding of the antibodies.
This finding indicates that a larger portion of the SBD is required for full binding of the antibodies in
immunoblotting experiments. Using various Hsp70 fragments, a region (aa 473-504) capable of
binding the antibodies in immunoblotting experiments was identified. N- or C-terminal deletions of
this region resulted in a complete loss of binding, indicating that the entire region is required. These
results suggest a discontinuous or partially conformational epitope. In a structural model of Hsp70,
this region, composed of 3 β-strands, is located adjacent to a β-strand containing the TKD sequence
(see Figure 3-17). Antibodies recognizing this larger epitope region might have been enriched during
the screening process on full-length Hsp70. Affinity data for binding of the Hsp70-specific antibodies
to this minimal region (FR18) and fragments also containing the TKD sequence are currently not
available. Such kinetic data would show if the TKD sequence is required to retain full antibody
binding activity. The identified epitope region is identical to the corresponding sequence of mouse
Hsp70, in line with the observation that cmHsp70.1, tumex IgG and humex IgG bind to human and
mouse Hsp70, e.g. as shown by immunoblotting experiments. In contrast, three residues (489, 491,
and 498) are different to the constitutively expressed Hsc70 (see also Figure 3-17). Because
cmHsp70.1 does not bind Hsc70, the conclusion is that these residues directly contribute to antigen
specificity.
Further studies have to be performed to analyze the cell binding activity of the chimeric and
humanized antibodies in comparison to the parental antibody and to demonstrate antitumor activity.
Friedrich and co-workers already demonstrated cell-binding activity of a chimeric Fab fragment
derived from cmHsp70.1 containing the identical CDRs as used in our humanized version (Friedrich et
al. 2010). Membrane-bound Hsp70 has no proliferative activity, hence antitumor effects of the antimHsp70 antibody will be primarily exerted through ADCC and CDC and not by blocking of mHsp70
(Gastpar et al. 2004). The capacity to mediate ADCC in vitro was already shown for cmHsp70.1
monoclonal antibody (Stangl et al. 2010). In this study, also in vivo homing of the fluorescent-labeled
antibody to CT26 mouse tumors and rapid internalization was shown. However, various studies with
cmHsp70.1 have shown that rather low amounts of Hsp70 are exposed in the membrane of tumor
cells (Hantschel et al. 2000). Thus, the parental and humanized antibody, exhibiting a nanomolar
affinity for Hsp70, may benefit from affinity maturation (Hoogenboom 2005; Presta 2006).
95
Kirstin A. Zettlitz
Discussion
Furthermore, engineering of the Fc region to exhibit an increased binding to FcγRIII and/or C1q, e.g.
through protein or glyco-engineering, might be a suitable option to improve the cytotoxic activity
and thus therapeutic efficacy (Presta 2008; Natsume et al. 2009). With the original as well as the
humanized sequences of the variable domains of cmHsp70.1 now available, such modifications can
be implemented in further developments of this anti-mHsp70 antibody as therapeutic reagent.
4.2 TNFR1-specific antibodies for the therapy of inflammatory diseases
4.2.1
Mono- and bivalent derivatives of humanized IZI-06.1
The second part of this thesis describes the generation of two monovalent albumin-fusion derivatives
(IZI-06.1 scFv-HSA and IZI-06.1 Fab-HSA) and the generation of an IgG1 derivative (ATROSAB) of the
humanized TNFR1-specific monoclonal antibody IZI-06.1 (Kontermann et al. 2008). The initially
reason for the generation of monovalent antibody formats was the anticipated advantageous for use
as a pure receptor antagonist that can block ligand binding to a receptor without cross-linking and
potentially activating the receptor. The fusion to human serum albumin should extend the terminal
half-life of the antibody fragments (scFv, Fab) (Kontermann 2009). Prolonged circulation times were
found for various albumin fusion proteins in various animals; like Fab and scFv-albumin fusion
proteins (Smith et al. 2001), rhIL2-rHSA (albuleukin (Melder et al. 2005)), and TNF-ligand (TNF, TRAIL,
TWEAK) fused to albumin (Muller et al. 2010). So far only few data are available on the
pharmacokinetic characteristics in humans. For an albumin-interferon-α fusion protein a prolonged
circulation time was reported in a phase I/II clinical trial (Balan et al. 2006).
The monovalent albumin-fusion antibodies showed some production and purification issues, such as
degradation and a tendency to aggregate. However, both constructs retain antigen binding even
though the affinity of IZI-06.1 scFv-HSA for human TNFR1-Fc was approximately 10-fold reduced
compared to IZI-06.1 scFv. A reason for the decreased affinity may be sterical hindrance by the
albumin. Further development of monovalent albumin-fusion derivatives based on IZI-06.1 will
require engineering for improved stability, solubility and production. A starting point could be the
peptide linker. Several studies have been made on the linker selection and suggest that the linker
conformation, flexibility, hydrophilicity and length (distance between protein domains) are important
not to disturb the functions of the linked domains (Crasto and Feng 2000).
The IgG format (IgG ATROSAB) was chosen because of its long half-life, established production and
increased binding due to bivalency. In order to avoid induction of antibody-mediated effector
functions, ATROSAB possesses an Fc-region deficient in activation of ADCC and CDC (Armour et al.
1999; Shields et al. 2001).
96
Kirstin A. Zettlitz
Discussion
Receptor-selective inhibition by ATROSAB and the parental mouse antibody resulted in blocking of
distinct signaling pathways of TNFR1, as shown by inhibition of TNF-mediated cell death and NF-κB
induced IL-6 and IL-8 release. Both cytokines are biomarkers of inflammation and are elevated e.g.
during episodes of active disease in RA (Terato et al. 1989). The antagonistic activity of the murine
H398 and the humanized monovalent Fab was described to be based on interference with ligand
binding (Thoma et al. 1990; Moosmayer et al. 1995; Kontermann et al. 2008). Using deletion mutants
the epitope recognized on TNFR1 was previously described to include the membrane-distal CRD1. By
using a domain swapping strategy for chimeric mouse/human TNFR1-Fc fusion proteins, it was now
shown that the epitope recognized by ATROSAB and H398 also includes subdomain A1 of CRD2, i.e.
the total epitope is covered by amino acids 1 to 70 in the N-terminal region of TNFR1. The finding
that also subdomain A1 of CRD2 is required for antibody binding hints toward sterical blockage as
cause for neutralization of TNF action. The structure of TNFR1 with bound TNF (Figure 3-39) shows
that the identified epitope region at least partially overlaps with the TNF binding site which is mainly
located in CRD2 and CRD3 (Banner et al. 1993). Additionally, site directed mutagenesis revealed that
residues Pro23 and Gln24 of subdomain A1 of CRD1 directly contribute to antigen and species
specificity. This is of interest as CRD1 is not directly involved in ligand binding (Banner et al. 1993) but
is critically involved in TNFR1 signaling. CRD1 controls high affinity ligand binding by stabilizing the
conformation of the subsequent CRD2 (Branschadel et al. 2009) and removal of CRD1 results in loss
of ligand binding (Chan et al. 2000; Siegel et al. 2000; Branschadel et al. 2009). In addition, CRD1
comprises a homophilic receptor/receptor interaction site, the pre-ligand-binding assembly domain
(PLAD) which is essential for generation of functional TNFR signal complexes (Chan et al. 2000;
Boschert et al. 2010). Hence, binding of ATROSAB to CRD1 could not only displace TNF by sterical
hindrance or by inducing a conformational change but could also interfere with homotypic PLAD
interactions, thereby blocking the formation of functional TNFR signal complexes.
ATROSAB showed a slightly reduced antagonistic activity compared to H398. This is probably not due
to altered affinity since affinities of both antibodies for recombinant human TNFR1 were similar as
determined by quartz crystal microbalance measurements and in flow cytometry measurements
using TNFR1-expressing cells. However, also in competition ELISA (Figure 3-30) 5 - 10-fold more
ATROSAB was required to compete against binding of H398 to human TNFR1-Fc. Furthermore, both
antibodies showed slight differences in binding to chimeric TNFR1-Fc in the epitope mapping
experiments, albeit this could be also caused by the different detection systems used. Currently, it
cannot be excluded that ATROSAB and H398 bind in a slightly different way or to a slightly different
area within the identified region (aa 1-70) containing the epitope. Further epitope mapping by site
97
Kirstin A. Zettlitz
Discussion
directed mutagenesis of exposed residues will provide insights into the exact localization of the
conformational epitope of ATROSAB and H398 and the mechanism of ligand blocking.
In absence of TNF, both antibodies (H398 and ATROSAB) also showed some minor agonistic activities
at low nanomolar concentrations. This marginal effect of the bivalent antibodies on the cytokine
release might be explained by cross-linking of receptors because monovalent Fab fragments of
ATROSAB and H398 did not exhibit any agonistic activities (not shown). However, IL-6 and IL-8 levels
increased only by 2 – 6 % compared to levels induced by TNF treatment. In these cell assays, IL-6
levels of up to 700 pg/ml were induced by TNF, comparable to IL-6 serum levels (400 - 600 pg/ml)
observed in rhesus monkeys suffering from collagen-induced arthritis (Vierboom et al. 2007).
ATROSAB showed no agonistic activity in experiments with human PBMCs (peripheral blood
mononuclear cells) and PMNs (polymorphonuclear neutrophils). PBMCs and PMNs express both TNF
receptors (Gehr et al. 1992) and showed cytokine production (IL-6, IL-8) or degranulation,
respectively, upon TNF-stimulation. It has been shown that antibodies with agonistic activity at
TNFR1 (e.g. Htr-9) mimicked the effect of TNF, while antagonistic antibody (e.g. TBP-2) lead to
inhibition of PMN response to TNF (Zeman et al. 1996). Menegazzi and co-workers investigated the
ability of H398 to influence superoxide (O2-) generation of PMN adherent to biologic surfaces.
Although H398 incubated alone (10 µg/ml equates to 62.5 nM) with adherent PMN, stimulated PMN
with a kinetic similar to TNF (10 µg/ml equates to 200 nM), no stimulation of superoxide production
by PMN in suspension was observed (Menegazzi et al. 1994). Recently, two groups described the
generation of antibodies antagonizing the activating receptor NKG2D on natural killer cells (Kwong et
al. 2008; Steigerwald et al. 2009). Both groups reported agonistic activity of the antibodies under
certain conditions, like immobilized antibody or antibody presented by cells bearing a high density of
FcγR, leading to extensively cross-linking of NKG2D. Because bivalent binding appeared neutral and
showed antagonistic effects in the presence of activating ligands, agonistic effects may be mediated
only by cross-linking of the antibodies. Therefore, non-depleting antibody formats like Fab, F(ab)2 or
antibodies with mutated Fc regions to diminish or eliminate Fc-receptor binding were suggested for
application of these antibodies in inflammatory or autoimmune diseases.
Importantly, binding of ATROSAB to rhesus TNF-R1 with a similar affinity as for human TNF-R1 could
be demonstrated, thus allowing for in vivo evaluation of ATROSAB in rhesus monkeys. The collageninduced arthritis (CIA) model is the recognized standard for potential RA therapeutics and could be
already reproducibly induced in rhesus macaques (Bakker et al. 1990). Because of the wellestablished proximity (physiological, anatomical, genetic, microbiological and immunological) with
humans, CIA in rhesus monkeys represents a very useful preclinical model for evaluation of safety
98
Kirstin A. Zettlitz
Discussion
and efficacy of novel therapies (t Hart et al. 2004) and enables the analysis of ATROSAB’s neutralizing
activity and safety in non human primates.
4.2.2
Optimization of IZI-06.1
Attempts towards affinity maturation of IZI-06.1 resulted in a pool of mutant scFv, thereof only
mutant scFv IG11 (library L2a, mutated in CDRH2) showed a two-fold increase in antigen-binding
affinity over the parental scFv IZI-06.1, which also translated into improved blocking activity of TNFmediated cytotoxicity in an in vitro cell assay. One mutant (scFv G3, library L1, mutated in
CDRH1/CDRL2) showed similar affinity and blocking activity compared to the wild type.
Affinity maturation of antibodies is often associated with increased in vitro biological activity
(Chowdhury and Wu 2005; Hoogenboom 2005) which may translate into increased in vivo activity.
Antibody affinity may be significantly improved by applying directed evolution technologies. These
technologies are characterized by several rounds of mutation, display, selection and amplification
steps. Selecting the appropriate technology to affinity mature an antibody depends upon the origin
of the antibody and the desired format (Wark and Hudson 2006). The library-construction strategies
vary from substitutions of specific selected residues within the CDR loops (rational design) to random
mutagenesis of the entire variable fragment (Fv). The several display technologies include phage
display, ribosome display (Luginbuhl et al. 2006), and yeast display (Graff et al. 2004). Thereof phage
display continues to be the most popular technology to screen antibody libraries and achieved
increased affinities from the nanomolar to picomolar range (Yang et al. 1995; Schier et al. 1996a).
While antibodies isolated from naïve or synthetic libraries have typically low dissociation constants
(KD) in the micromolar range (Yau et al. 2005), not sufficient for therapeutic applications, antibodies
generated in vivo can reach affinities up to 100 pM (Lowe and Jermutus 2004). The mouse mAb
H398, generated by immunization of mice with purified TNF receptor material (Thoma et al. 1990)
has a quite high huTNFR1-binding affinity of approximately 200 pM. This affinity could be retained for
the humanized version ATROSAB (approximately 350 pM), yet it showed weaker blocking activity and
might benefit from affinity maturation. Furthermore, the antibody has to compete with soluble TNF
for binding to TNFR1, which has a high affinity of approximately 100 pM (Kontermann et al. 2008;
Boschert et al. 2010).
The CDRs, in particular CDRs3, which contain the most extensive sequence variation, are commonly
targeted for in vitro mutagenesis. Because mAb H398 has been matured in vivo and already shows
good affinity, it was assumed that CDRH3 and CDRL3, which contribute most of the binding energy,
may not be amenable for further mutations. The antigen binding site of antibodies that recognize
99
Kirstin A. Zettlitz
Discussion
large protein antigens are generally planar (Jones and Thornton 1996), and large antigens often
contact antibody residues at the edge of the combining site and interact with more apical portions of
the CDR loops (MacCallum et al. 1996). In the ideal case where high-resolution antibody structure or,
preferably, antibody-antigen complex structures are available, determination of contact residues is
straightforward and this information can be applied to guide the maturation process (Clark et al.
2006). However, also a molecular model of the antibody, like used in this study, can provide a useful
starting point for predicting the site of useful mutations, because residues that make contact with
the antigen are necessarily those at the surface. Site directed mutagenesis was used to introduce
mutations into the CDRs 1 and 2 of both light and heavy chain, and targeted exposed residues. It is
difficult to make libraries greater than 107 – 108 clones, which limits the number of amino acids to
diversify as well as the extent of randomization. One of the main problems associated with affinity
maturation by phage display is that of library completeness. Experience with phage display libraries
supports the prediction that the probability of the identification of higher-affinity clones increases
with the size and functionality of the display library (the number of clones that is displayed with
sufficient efficiency to be potentially selectable) (Perelson and Oster 1979; Bradbury and Marks
2004). Therefore, four different libraries were generated with mutations either in CDRH1/CDRL2 (L1),
CDRH2/CDRL1 (L2), CDRH2 (L2a) or CDRL1 (L2b) and selected in parallel for binding to both human
and mouse TNFR1-Fc. Selection and screening of library L2 (CDRH2/CDRL1) did not result in
identification of clones binding to human TNFR1, presumably randomized positions affected one or
more critical residues, which were not present in the correct composition in library 2 covering only a
small fraction of all possible combinations. Selection of libraries L1, L2a and L2b on the other hand
yielded a number of mutant scFv showing binding to human TNFR1. Unfortunately, none of the
selections on mouse TNFR1-Fc resulted in cross-reactive clones, indicating that mutations at the
chosen positions are not able to confer binding to mouse TNFR1. Probably, the sequence-relatedness
between mouse and human TNFR1 is not high enough and the epitope is not conserved.
Two approaches have been used for the selection of higher affinity clones from diversified libraries:
Selection with immobilized antigen and selection with antigen in solution. None of the mutants
selected on immobilized antigen in immunotubes was predominant, although sequence analysis
revealed preferences for certain amino acid residues at the mutated positions. ScFv from each
library, representing the preferred amino acid, were bacterially produced and the antigen-binding
affinity was analyzed. None of the mutants showed increased affinity, only scFv L1_G3 showed
similar affinity and blocking activity compared to wild type scFv IZI-06.1. This may be due to the
selection method with immobilized antigen, which is likely to result in the selection of phage
displaying multiple copies of the scFv on the surface or phage displaying dimeric scFv which have a
100
Kirstin A. Zettlitz
Discussion
higher functional affinity (avidity) but the monovalent binding constant is no higher than the wild
type (Schier et al. 1996b). This is important when selecting scFv libraries, where it is known that the
scFv can spontaneously dimerize, like IZI-06.1 scFv does (see SEC analysis,Figure 3-18). Interestingly,
the only mutant (Lib2a_IG11) showing at least two-fold higher affinity compared to IZI-06.1 scFv,
resulted from six rounds of equilibrium selection. In this method phage were incubated with soluble
biotinylated antigen at decreasing concentrations (ideally below the equilibrium constant) and bound
phage were recovered using streptavidin-coated magnetic beads. It is described that reduction of the
antigen concentration leads to the selection of higher affinity scFv (Boder and Wittrup 1998). The
higher affinity of clone L2a_IG11 resulted from a 1.6-fold increase in kon and a 3-fold decrease in koff.
Increases in affinity achieved by display technology are typically due to reductions in dissociation
rate. But also increases in association rate have been reported for antibodies that have undergone
affinity maturation using display technology and targeted screening or selection strategies (Razai et
al. 2005; Wu et al. 2005).
Beneficial mutations from independently randomized CDRs can be combined in single clones and
often further increase affinity in an additive or synergistic manner (Wells 1990). Although this
strategy has been successfully employed for optimization of several antibodies, not all mutations
have been additive (Yang et al. 1995; Schier et al. 1996c). This is no surprise since the CDRs lie in
close spatial proximity and many residues pack against one another. In this work, the combination of
the CDRs of the best candidates from immunotube selections in two scFv (kze1 and kze2) resulted in
complete loss of antigen binding. This is probably due to unfavorable combination of CDRL1 (clone
L2b_F3) and CDRH2 (clone L2a_A5), which both had lower affinities than the parental scFv, with
CDRH1 and CDRL2 (clone L1_G3) which had similar affinity. Changing the CDRH2 to the sequence of
mutant L2a_IG11 (scFv kze3) restored antigen binding and indicates the importance of CDRH2.
However, no blocking activity was seen for scFv kze3. Mutating CDRL1 back to wild type results in a
scFv (scFv kze4) with blocking activity, although the activity is weaker compared to scFv IZI-06.1 and
L2a_IG11.
The results for affinity maturation suggest that improvement of antigen-binding by mutation of CDR1
and 2 is possible and for CDRH2 and CDRL1 a strong impact on antigen binding was shown.
Furthermore, the selection method was crucial for identification of high affinity binders, for that
reason, selection of libraries L1 and L2b should be repeated using selection with soluble antigen.
Other selection methods should also be considered, like off-rate selection, that is selections based on
binding kinetics, in which the phage are allowed to saturate with the labeled antigen before a large
molar excess of unlabeled antigen is added to the mix for a given amount of time. The majority of the
101
Kirstin A. Zettlitz
Discussion
bound wild type clones or clones with lower affinity will dissociate while the improved mutants
remain bound on the labeled antigen (Hawkins et al. 1992). This method selects for mutants with a
slower off-rate (koff) which is typically the major kinetic mechanism resulting in higher affinity when V
genes are mutated (Marks et al. 1992). Additionally, the screening process could be optimized by
using quartz crystal microbalance measurements for identifying the best candidates directly based on
the off rate from unpurified samples.
A sequential mutation strategy based on clone scFv L2a_IG11 might be most promising, given that
combination of separately optimized CDRs had negative effects on binding. Also the mutation of
CDRH3 and CDRL3 should be considered, these CDRs generally make more extensive contact with the
antigen. However, it is not guaranteed that substituting any of the contact residues will improve
binding. Successful affinity maturation may also be achieved using other methods like CDR walking
(Barbas et al. 1994), hot-spot mutagenesis (Ho et al. 2005) or random mutagenesis of the complete
variable domains by error-prone PCR (Luginbuhl et al. 2006), chain shuffling or using in vivo sytems
like “mutator” E. coli strains or mammalian cells (B cells). Randomization and selection studies often
yields substitutes residues that are not in contact with the antigen (Valjakka et al. 2002). Hence, it is
extremely complicated to determine which residues make up the binding site, which of them can be
improved, and which peripheral residues should also be considered. Alternatively, randomization of
the antigen binding site of IZI-06.1 and selection to bias an epitope shift could result in antibodies
with improved neutralization activity.
In conclusion, the results of this study demonstrate that a number of prerequisites for the
development of ATROSAB as a therapeutic are fulfilled, like lowest possible immunogenicity, high
affinity and potency, high stability, cross-reactivity with relevant animals and defined biological
activity. The next step would be the evaluation of the in vivo efficacy in a relevant disease model.
Additionally, the results indicate that further engineering of ATROSAB offers a number of benefits for
its therapeutic efficacy, and both affinity maturation and alternative antibody formats should be
taken into consideration. TNFR1-selective antagonist, such as ATROSAB, will permit new therapeutic
options for diseases where anti-TNF therapeutics failed or even exacerbate disease progression,
including multiple sclerosis, congestive heart failure, metabolic diseases (type II diabetes), cytokine
release syndrome, septic shock, acute (stroke) and chronic (Alzheimer and Parkinson disease)
neurodegenerative diseases. ATROSAB could be an especially useful therapeutic alternative in
diseases already known to clinically respond to anti-TNF treatment and particularly in those diseases
where specific blockage of TNFR1 and maintenance of TNFR2 function appears as a promising
therapeutic approach.
102
Kirstin A. Zettlitz
References
5 References
Abhinandan, K. R. and Martin, A. C. (2007). "Analyzing the "degree of humanness" of antibody
sequences." J Mol Biol 369(3): 852-862.
Adams, G. P., Schier, R., Marshall, K., Wolf, E. J., McCall, A. M., Marks, J. D. and Weiner, L. M. (1998).
"Increased affinity leads to improved selective tumor delivery of single-chain Fv antibodies."
Cancer Res 58(3): 485-490.
Adams, G. P., Schier, R., McCall, A. M., Simmons, H. H., Horak, E. M., Alpaugh, R. K., Marks, J. D. and
Weiner, L. M. (2001). "High affinity restricts the localization and tumor penetration of singlechain fv antibody molecules." Cancer Res 61(12): 4750-4755.
Adams, G. P. and Weiner, L. M. (2005). "Monoclonal antibody therapy of cancer." Nat Biotechnol
23(9): 1147-1157.
Aggarwal, S. (2009). "What's fueling the biotech engine--2008." Nat Biotechnol 27(11): 987-993.
Al-Lazikani, B., Lesk, A. M. and Chothia, C. (1997). "Standard conformations for the canonical
structures of immunoglobulins." J Mol Biol 273(4): 927-948.
Alegre, M. L., Peterson, L. J., Xu, D., Sattar, H. A., Jeyarajah, D. R., Kowalkowski, K., Thistlethwaite, J.
R., Zivin, R. A., Jolliffe, L. and Bluestone, J. A. (1994). "A non-activating "humanized" anti-CD3
monoclonal antibody retains immunosuppressive properties in vivo." Transplantation 57(11):
1537-1543.
Almagro, J. C. and Fransson, J. (2008). "Humanization of antibodies." Front Biosci 13: 1619-1633.
Armour, K. L., Clark, M. R., Hadley, A. G. and Williamson, L. M. (1999). "Recombinant human IgG
molecules lacking Fcgamma receptor I binding and monocyte triggering activities." Eur J
Immunol 29(8): 2613-2624.
Baert, F., Noman, M., Vermeire, S., Van Assche, G., G, D. H., Carbonez, A. and Rutgeerts, P. (2003).
"Influence of immunogenicity on the long-term efficacy of infliximab in Crohn's disease." N
Engl J Med 348(7): 601-608.
Bakker, N. P., van Erck, M. G., Zurcher, C., Faaber, P., Lemmens, A., Hazenberg, M., Bontrop, R. E. and
Jonker, M. (1990). "Experimental immune mediated arthritis in rhesus monkeys. A model for
human rheumatoid arthritis?" Rheumatol Int 10(1): 21-29.
Balan, V., Nelson, D. R., Sulkowski, M. S., Everson, G. T., Lambiase, L. R., Wiesner, R. H., Dickson, R. C.,
Post, A. B., Redfield, R. R., Davis, G. L., Neumann, A. U., Osborn, B. L., Freimuth, W. W. and
Subramanian, G. M. (2006). "A Phase I/II study evaluating escalating doses of recombinant
human albumin-interferon-alpha fusion protein in chronic hepatitis C patients who have
failed previous interferon-alpha-based therapy." Antivir Ther 11(1): 35-45.
Banner, D. W., D'Arcy, A., Janes, W., Gentz, R., Schoenfeld, H. J., Broger, C., Loetscher, H. and
Lesslauer, W. (1993). "Crystal structure of the soluble human 55 kd TNF receptor-human TNF
beta complex: implications for TNF receptor activation." Cell 73(3): 431-445.
Barbas, C. F., 3rd, Hu, D., Dunlop, N., Sawyer, L., Cababa, D., Hendry, R. M., Nara, P. L. and Burton, D.
R. (1994). "In vitro evolution of a neutralizing human antibody to human immunodeficiency
virus type 1 to enhance affinity and broaden strain cross-reactivity." Proc Natl Acad Sci U S A
91(9): 3809-3813.
Beck, A., Wurch, T., Bailly, C. and Corvaia, N. (2010). "Strategies and challenges for the next
generation of therapeutic antibodies." Nat Rev Immunol 10(5): 345-352.
Beck, A., Wurch, T. and Corvaia, N. (2008). "Therapeutic antibodies and derivatives: from the bench
to the clinic." Curr Pharm Biotechnol 9(6): 421-422.
Benedict, C. A., MacKrell, A. J. and Anderson, W. F. (1997). "Determination of the binding affinity of
an anti-CD34 single-chain antibody using a novel, flow cytometry based assay." J Immunol
Methods 201(2): 223-231.
Berard, F., Blanco, P., Davoust, J., Neidhart-Berard, E. M., Nouri-Shirazi, M., Taquet, N., Rimoldi, D.,
Cerottini, J. C., Banchereau, J. and Palucka, A. K. (2000). "Cross-priming of naive CD8 T cells
103
Kirstin A. Zettlitz
References
against melanoma antigens using dendritic cells loaded with killed allogeneic melanoma
cells." J Exp Med 192(11): 1535-1544.
Bibeau, F., Lopez-Crapez, E., Di Fiore, F., Thezenas, S., Ychou, M., Blanchard, F., Lamy, A., PenaultLlorca, F., Frebourg, T., Michel, P., Sabourin, J. C. and Boissiere-Michot, F. (2009). "Impact of
Fc{gamma}RIIa-Fc{gamma}RIIIa polymorphisms and KRAS mutations on the clinical outcome
of patients with metastatic colorectal cancer treated with cetuximab plus irinotecan." J Clin
Oncol 27(7): 1122-1129.
Black, R. A., Rauch, C. T., Kozlosky, C. J., Peschon, J. J., Slack, J. L., Wolfson, M. F., Castner, B. J.,
Stocking, K. L., Reddy, P., Srinivasan, S., Nelson, N., Boiani, N., Schooley, K. A., Gerhart, M.,
Davis, R., Fitzner, J. N., Johnson, R. S., Paxton, R. J., March, C. J. and Cerretti, D. P. (1997). "A
metalloproteinase disintegrin that releases tumour-necrosis factor-alpha from cells." Nature
385(6618): 729-733.
Boder, E. T. and Wittrup, K. D. (1998). "Optimal screening of surface-displayed polypeptide libraries."
Biotechnol Prog 14(1): 55-62.
Bolt, S., Routledge, E., Lloyd, I., Chatenoud, L., Pope, H., Gorman, S. D., Clark, M. and Waldmann, H.
(1993). "The generation of a humanized, non-mitogenic CD3 monoclonal antibody which
retains in vitro immunosuppressive properties." Eur J Immunol 23(2): 403-411.
Bongartz, T., Sutton, A. J., Sweeting, M. J., Buchan, I., Matteson, E. L. and Montori, V. (2006). "AntiTNF antibody therapy in rheumatoid arthritis and the risk of serious infections and
malignancies: systematic review and meta-analysis of rare harmful effects in randomized
controlled trials." Jama 295(19): 2275-2285.
Boschert, V., Krippner-Heidenreich, A., Branschadel, M., Tepperink, J., Aird, A. and Scheurich, P.
(2010). "Single chain TNF derivatives with individually mutated receptor binding sites reveal
differential stoichiometry of ligand receptor complex formation for TNFR1 and TNFR2." Cell
Signal.
Bothmann, H. and Pluckthun, A. (2000). "The periplasmic Escherichia coli peptidylprolyl cis,transisomerase FkpA. I. Increased functional expression of antibody fragments with and without
cis-prolines." J Biol Chem 275(22): 17100-17105.
Botzler, C., Li, G., Issels, R. D. and Multhoff, G. (1998). "Definition of extracellular localized epitopes
of Hsp70 involved in an NK immune response." Cell Stress Chaperones 3(1): 6-11.
Bradbury, A. R. and Marks, J. D. (2004). "Antibodies from phage antibody libraries." J Immunol
Methods 290(1-2): 29-49.
Branschadel, M., Aird, A., Zappe, A., Tietz, C., Krippner-Heidenreich, A. and Scheurich, P. (2009).
"Dual function of cysteine rich domain (CRD) 1 of TNF receptor type 1: conformational
stabilization of CRD2 and control of receptor responsiveness." Cell Signal 22(3): 404-414.
Calderwood, S. K., Theriault, J. R. and Gong, J. (2005). "Message in a bottle: role of the 70-kDa heat
shock protein family in anti-tumor immunity." Eur J Immunol 35(9): 2518-2527.
Carter, P. (2001). "Improving the efficacy of antibody-based cancer therapies." Nat Rev Cancer 1(2):
118-129.
Carter, P., Bedouelle, H. and Winter, G. (1985). "Improved oligonucleotide site-directed mutagenesis
using M13 vectors." Nucleic Acids Res 13(12): 4431-4443.
Carter, P., Presta, L., Gorman, C. M., Ridgway, J. B., Henner, D., Wong, W. L., Rowland, A. M., Kotts,
C., Carver, M. E. and Shepard, H. M. (1992). "Humanization of an anti-p185HER2 antibody for
human cancer therapy." Proc Natl Acad Sci U S A 89(10): 4285-4289.
Carter, P., Smith, L. and Ryan, M. (2004). "Identification and validation of cell surface antigens for
antibody targeting in oncology." Endocr Relat Cancer 11(4): 659-687.
Carter, P. J. (2006). "Potent antibody therapeutics by design." Nat Rev Immunol 6(5): 343-357.
Cartron, G., Dacheux, L., Salles, G., Solal-Celigny, P., Bardos, P., Colombat, P. and Watier, H. (2002).
"Therapeutic activity of humanized anti-CD20 monoclonal antibody and polymorphism in IgG
Fc receptor FcgammaRIIIa gene." Blood 99(3): 754-758.
104
Kirstin A. Zettlitz
References
Cartron, G., Watier, H., Golay, J. and Solal-Celigny, P. (2004). "From the bench to the bedside: ways to
improve rituximab efficacy." Blood 104(9): 2635-2642.
Chan, A. C. and Carter, P. J. (2010). "Therapeutic antibodies for autoimmunity and inflammation." Nat
Rev Immunol 10(5): 301-316.
Chan, F. K., Chun, H. J., Zheng, L., Siegel, R. M., Bui, K. L. and Lenardo, M. J. (2000). "A domain in TNF
receptors that mediates ligand-independent receptor assembly and signaling." Science
288(5475): 2351-2354.
Chirino, A. J., Ary, M. L. and Marshall, S. A. (2004). "Minimizing the immunogenicity of protein
therapeutics." Drug Discov Today 9(2): 82-90.
Chowdhury, P. S. and Wu, H. (2005). "Tailor-made antibody therapeutics." Methods 36(1): 11-24.
Choy, E. H., Hazleman, B., Smith, M., Moss, K., Lisi, L., Scott, D. G., Patel, J., Sopwith, M. and Isenberg,
D. A. (2002). "Efficacy of a novel PEGylated humanized anti-TNF fragment (CDP870) in
patients with rheumatoid arthritis: a phase II double-blinded, randomized, dose-escalating
trial." Rheumatology (Oxford) 41(10): 1133-1137.
Chung, C. H., Mirakhur, B., Chan, E., Le, Q. T., Berlin, J., Morse, M., Murphy, B. A., Satinover, S. M.,
Hosen, J., Mauro, D., Slebos, R. J., Zhou, Q., Gold, D., Hatley, T., Hicklin, D. J. and Platts-Mills,
T. A. (2008). "Cetuximab-induced anaphylaxis and IgE specific for galactose-alpha-1,3galactose." N Engl J Med 358(11): 1109-1117.
Clark, L. A., Boriack-Sjodin, P. A., Eldredge, J., Fitch, C., Friedman, B., Hanf, K. J., Jarpe, M., Liparoto, S.
F., Li, Y., Lugovskoy, A., Miller, S., Rushe, M., Sherman, W., Simon, K. and Van Vlijmen, H.
(2006). "Affinity enhancement of an in vivo matured therapeutic antibody using structurebased computational design." Protein Sci 15(5): 949-960.
Clynes, R. A., Towers, T. L., Presta, L. G. and Ravetch, J. V. (2000). "Inhibitory Fc receptors modulate in
vivo cytoxicity against tumor targets." Nat Med 6(4): 443-446.
Co, M. S., Avdalovic, N. M., Caron, P. C., Avdalovic, M. V., Scheinberg, D. A. and Queen, C. (1992).
"Chimeric and humanized antibodies with specificity for the CD33 antigen." J Immunol
148(4): 1149-1154.
Co, M. S., Scheinberg, D. A., Avdalovic, N. M., McGraw, K., Vasquez, M., Caron, P. C. and Queen, C.
(1993). "Genetically engineered deglycosylation of the variable domain increases the affinity
of an anti-CD33 monoclonal antibody." Mol Immunol 30(15): 1361-1367.
Coiffier, B., Lepretre, S., Pedersen, L. M., Gadeberg, O., Fredriksen, H., van Oers, M. H., Wooldridge,
J., Kloczko, J., Holowiecki, J., Hellmann, A., Walewski, J., Flensburg, M., Petersen, J. and
Robak, T. (2008). "Safety and efficacy of ofatumumab, a fully human monoclonal anti-CD20
antibody, in patients with relapsed or refractory B-cell chronic lymphocytic leukemia: a phase
1-2 study." Blood 111(3): 1094-1100.
Cragg, M. S. and Glennie, M. J. (2004). "Antibody specificity controls in vivo effector mechanisms of
anti-CD20 reagents." Blood 103(7): 2738-2743.
Crameri, A., Cwirla, S. and Stemmer, W. P. (1996). "Construction and evolution of antibody-phage
libraries by DNA shuffling." Nat Med 2(1): 100-102.
Crasto, C. J. and Feng, J. A. (2000). "LINKER: a program to generate linker sequences for fusion
proteins." Protein Eng 13(5): 309-312.
Dall'Acqua, W. F., Kiener, P. A. and Wu, H. (2006). "Properties of human IgG1s engineered for
enhanced binding to the neonatal Fc receptor (FcRn)." J Biol Chem 281(33): 23514-23524.
Damschroder, M. M., Widjaja, L., Gill, P. S., Krasnoperov, V., Jiang, W., Dall'Acqua, W. F. and Wu, H.
(2007). "Framework shuffling of antibodies to reduce immunogenicity and manipulate
functional and biophysical properties." Mol Immunol 44(11): 3049-3060.
Desai, S. B. and Furst, D. E. (2006). "Problems encountered during anti-tumour necrosis factor
therapy." Best Pract Res Clin Rheumatol 20(4): 757-790.
Di Gaetano, N., Cittera, E., Nota, R., Vecchi, A., Grieco, V., Scanziani, E., Botto, M., Introna, M. and
Golay, J. (2003). "Complement activation determines the therapeutic activity of rituximab in
vivo." J Immunol 171(3): 1581-1587.
105
Kirstin A. Zettlitz
References
Duncan, A. R. and Winter, G. (1988). "The binding site for C1q on IgG." Nature 332(6166): 738-740.
Duncan, A. R., Woof, J. M., Partridge, L. J., Burton, D. R. and Winter, G. (1988). "Localization of the
binding site for the human high-affinity Fc receptor on IgG." Nature 332(6164): 563-564.
Dunkelberger, J. R. and Song, W. C. (2010). "Complement and its role in innate and adaptive immune
responses." Cell Res 20(1): 34-50.
Ehrlich, P. (1906). Collected studies on immunity, Wiley.
Ewert, S., Honegger, A. and Pluckthun, A. (2004). "Stability improvement of antibodies for
extracellular and intracellular applications: CDR grafting to stable frameworks and structurebased framework engineering." Methods 34(2): 184-199.
Farkas, B., Hantschel, M., Magyarlaki, M., Becker, B., Scherer, K., Landthaler, M., Pfister, K.,
Gehrmann, M., Gross, C., Mackensen, A. and Multhoff, G. (2003). "Heat shock protein 70
membrane expression and melanoma-associated marker phenotype in primary and
metastatic melanoma." Melanoma Res 13(2): 147-152.
Figini, M., Marks, J. D., Winter, G. and Griffiths, A. D. (1994). "In vitro assembly of repertoires of
antibody chains on the surface of phage by renaturation." J Mol Biol 239(1): 68-78.
Foote, J. and Winter, G. (1992). "Antibody framework residues affecting the conformation of the
hypervariable loops." J Mol Biol 224(2): 487-499.
Fredericks, Z. L., Forte, C., Capuano, I. V., Zhou, H., Vanden Bos, T. and Carter, P. (2004).
"Identification of potent human anti-IL-1RI antagonist antibodies." Protein Eng Des Sel 17(1):
95-106.
Friedrich, L., Stangl, S., Hahne, H., Kuster, B., Kohler, P., Multhoff, G. and Skerra, A. (2010). "Bacterial
production and functional characterization of the Fab fragment of the murine IgG1/lambda
monoclonal antibody cmHsp70.1, a reagent for tumour diagnostics." Protein Eng Des Sel
23(4): 161-168.
Fujimori, K., Covell, D. G., Fletcher, J. E. and Weinstein, J. N. (1989). "Modeling analysis of the global
and microscopic distribution of immunoglobulin G, F(ab')2, and Fab in tumors." Cancer Res
49(20): 5656-5663.
Gastpar, R., Gehrmann, M., Bausero, M. A., Asea, A., Gross, C., Schroeder, J. A. and Multhoff, G.
(2005). "Heat shock protein 70 surface-positive tumor exosomes stimulate migratory and
cytolytic activity of natural killer cells." Cancer Res 65(12): 5238-5247.
Gastpar, R., Gross, C., Rossbacher, L., Ellwart, J., Riegger, J. and Multhoff, G. (2004). "The cell surfacelocalized heat shock protein 70 epitope TKD induces migration and cytolytic activity
selectively in human NK cells." J Immunol 172(2): 972-980.
Gehr, G., Gentz, R., Brockhaus, M., Loetscher, H. and Lesslauer, W. (1992). "Both tumor necrosis
factor receptor types mediate proliferative signals in human mononuclear cell activation." J
Immunol 149(3): 911-917.
Gehrmann, M., Brunner, M., Pfister, K., Reichle, A., Kremmer, E. and Multhoff, G. (2004). "Differential
up-regulation of cytosolic and membrane-bound heat shock protein 70 in tumor cells by antiinflammatory drugs." Clin Cancer Res 10(10): 3354-3364.
Gehrmann, M., Schmetzer, H., Eissner, G., Haferlach, T., Hiddemann, W. and Multhoff, G. (2003).
"Membrane-bound heat shock protein 70 (Hsp70) in acute myeloid leukemia: a tumor
specific recognition structure for the cytolytic activity of autologous NK cells." Haematologica
88(4): 474-476.
Ghetie, V. and Ward, E. S. (2002). "Transcytosis and catabolism of antibody." Immunol Res 25(2): 97113.
Gonzales, N. R., De Pascalis, R., Schlom, J. and Kashmiri, S. V. (2005). "Minimizing the immunogenicity
of antibodies for clinical application." Tumour Biol 26(1): 31-43.
Graff, C. P., Chester, K., Begent, R. and Wittrup, K. D. (2004). "Directed evolution of an anticarcinoembryonic antigen scFv with a 4-day monovalent dissociation half-time at 37 degrees
C." Protein Eng Des Sel 17(4): 293-304.
106
Kirstin A. Zettlitz
References
Green, L. L., Hardy, M. C., Maynard-Currie, C. E., Tsuda, H., Louie, D. M., Mendez, M. J., Abderrahim,
H., Noguchi, M., Smith, D. H., Zeng, Y. and et al. (1994). "Antigen-specific human monoclonal
antibodies from mice engineered with human Ig heavy and light chain YACs." Nat Genet 7(1):
13-21.
Griffiths, A. D., Williams, S. C., Hartley, O., Tomlinson, I. M., Waterhouse, P., Crosby, W. L.,
Kontermann, R. E., Jones, P. T., Low, N. M., Allison, T. J. and et al. (1994). "Isolation of high
affinity human antibodies directly from large synthetic repertoires." Embo J 13(14): 32453260.
Gross, C., Koelch, W., DeMaio, A., Arispe, N. and Multhoff, G. (2003). "Cell surface-bound heat shock
protein 70 (Hsp70) mediates perforin-independent apoptosis by specific binding and uptake
of granzyme B." J Biol Chem 278(42): 41173-41181.
Hanauer, S. B., Feagan, B. G., Lichtenstein, G. R., Mayer, L. F., Schreiber, S., Colombel, J. F.,
Rachmilewitz, D., Wolf, D. C., Olson, A., Bao, W. and Rutgeerts, P. (2002). "Maintenance
infliximab for Crohn's disease: the ACCENT I randomised trial." Lancet 359(9317): 1541-1549.
Hantschel, M., Pfister, K., Jordan, A., Scholz, R., Andreesen, R., Schmitz, G., Schmetzer, H.,
Hiddemann, W. and Multhoff, G. (2000). "Hsp70 plasma membrane expression on primary
tumor biopsy material and bone marrow of leukemic patients." Cell Stress Chaperones 5(5):
438-442.
Hawkins, R. E., Russell, S. J. and Winter, G. (1992). "Selection of phage antibodies by binding affinity.
Mimicking affinity maturation." J Mol Biol 226(3): 889-896.
Hinton, P. R., Johlfs, M. G., Xiong, J. M., Hanestad, K., Ong, K. C., Bullock, C., Keller, S., Tang, M. T.,
Tso, J. Y., Vasquez, M. and Tsurushita, N. (2004). "Engineered human IgG antibodies with
longer serum half-lives in primates." J Biol Chem 279(8): 6213-6216.
Ho, M., Kreitman, R. J., Onda, M. and Pastan, I. (2005). "In vitro antibody evolution targeting germline
hot spots to increase activity of an anti-CD22 immunotoxin." J Biol Chem 280(1): 607-617.
Honegger, A. (2008). "Engineering antibodies for stability and efficient folding." Handb Exp
Pharmacol(181): 47-68.
Hoogenboom, H. R. (2005). "Selecting and screening recombinant antibody libraries." Nat Biotechnol
23(9): 1105-1116.
Hwang, W. Y. and Foote, J. (2005). "Immunogenicity of engineered antibodies." Methods 36(1): 3-10.
Idusogie, E. E., Presta, L. G., Gazzano-Santoro, H., Totpal, K., Wong, P. Y., Ultsch, M., Meng, Y. G. and
Mulkerrin, M. G. (2000). "Mapping of the C1q binding site on rituxan, a chimeric antibody
with a human IgG1 Fc." J Immunol 164(8): 4178-4184.
Idusogie, E. E., Wong, P. Y., Presta, L. G., Gazzano-Santoro, H., Totpal, K., Ultsch, M. and Mulkerrin,
M. G. (2001). "Engineered antibodies with increased activity to recruit complement." J
Immunol 166(4): 2571-2575.
Jacquemin, M., Radcliffe, C. M., Lavend'homme, R., Wormald, M. R., Vanderelst, L., Wallays, G.,
Dewaele, J., Collen, D., Vermylen, J., Dwek, R. A., Saint-Remy, J. M., Rudd, P. M. and
Dewerchin, M. (2006). "Variable region heavy chain glycosylation determines the
anticoagulant activity of a factor VIII antibody." J Thromb Haemost 4(5): 1047-1055.
Jefferis, R. (2005). "Glycosylation of recombinant antibody therapeutics." Biotechnol Prog 21(1): 1116.
Jefferis, R. (2009a). "Glycosylation as a strategy to improve antibody-based therapeutics." Nat Rev
Drug Discov 8(3): 226-234.
Jefferis, R. (2009b). "Glycosylation of antibody therapeutics: optimisation for purpose." Methods Mol
Biol 483: 223-238.
Jones, P. T., Dear, P. H., Foote, J., Neuberger, M. S. and Winter, G. (1986). "Replacing the
complementarity-determining regions in a human antibody with those from a mouse."
Nature 321(6069): 522-525.
Jones, S. and Thornton, J. M. (1996). "Principles of protein-protein interactions." Proc Natl Acad Sci U
S A 93(1): 13-20.
107
Kirstin A. Zettlitz
References
Kleinjung, T., Arndt, O., Feldmann, H. J., Bockmuhl, U., Gehrmann, M., Zilch, T., Pfister, K.,
Schonberger, J., Marienhagen, J., Eilles, C., Rossbacher, L. and Multhoff, G. (2003). "Heat
shock protein 70 (Hsp70) membrane expression on head-and-neck cancer biopsy-a target for
natural killer (NK) cells." Int J Radiat Oncol Biol Phys 57(3): 820-826.
Köhler, G. and Milstein, C. (1975). "Continuous cultures of fused cells secreting antibody of
predefined specificity." Nature 256(5517): 495-497.
Koike, C., Uddin, M., Wildman, D. E., Gray, E. A., Trucco, M., Starzl, T. E. and Goodman, M. (2007).
"Functionally important glycosyltransferase gain and loss during catarrhine primate
emergence." Proc Natl Acad Sci U S A 104(2): 559-564.
Kontermann, R. E. (2009). "Strategies to extend plasma half-lives of recombinant antibodies."
BioDrugs 23(2): 93-109.
Kontermann, R. E., Martineau, P., Cummings, C. E., Karpas, A., Allen, D., Derbyshire, E. and Winter, G.
(1997). "Enzyme immunoassays using bispecific diabodies." Immunotechnology 3(2): 137144.
Kontermann, R. E., Munkel, S., Neumeyer, J., Muller, D., Branschadel, M., Scheurich, P. and
Pfizenmaier, K. (2008). "A humanized tumor necrosis factor receptor 1 (TNFR1)-specific
antagonistic antibody for selective inhibition of tumor necrosis factor (TNF) action." J
Immunother 31(3): 225-234.
Kontermann, R. E., Scheurich, P. and Pfizenmaier, K. (2009). "Antagonists of TNF action - clinical
experience and new developments." Expert Opin. Drug Discov. 4(3): 279-292.
Krause, S. W., Gastpar, R., Andreesen, R., Gross, C., Ullrich, H., Thonigs, G., Pfister, K. and Multhoff, G.
(2004). "Treatment of colon and lung cancer patients with ex vivo heat shock protein 70peptide-activated, autologous natural killer cells: a clinical phase i trial." Clin Cancer Res
10(11): 3699-3707.
Krippner-Heidenreich, A., Tubing, F., Bryde, S., Willi, S., Zimmermann, G. and Scheurich, P. (2002).
"Control of receptor-induced signaling complex formation by the kinetics of ligand/receptor
interaction." J Biol Chem 277(46): 44155-44163.
Kruppa, G., Thoma, B., Machleidt, T., Wiegmann, K. and Kronke, M. (1992). "Inhibition of tumor
necrosis factor (TNF)-mediated NF-kappa B activation by selective blockade of the human 55kDa TNF receptor." J Immunol 148(10): 3152-3157.
Kubota, T., Niwa, R., Satoh, M., Akinaga, S., Shitara, K. and Hanai, N. (2009). "Engineered therapeutic
antibodies with improved effector functions." Cancer Sci 100(9): 1566-1572.
Kwong, K. Y., Baskar, S., Zhang, H., Mackall, C. L. and Rader, C. (2008). "Generation, affinity
maturation, and characterization of a human anti-human NKG2D monoclonal antibody with
dual antagonistic and agonistic activity." J Mol Biol 384(5): 1143-1156.
Labrijn, A. F., Aalberse, R. C. and Schuurman, J. (2008). "When binding is enough: nonactivating
antibody formats." Curr Opin Immunol 20(4): 479-485.
Laemmli, U. K. (1970). "Cleavage of structural proteins during the assembly of the head of
bacteriophage T4." Nature 227(5259): 680-685.
Lazar, G. A., Dang, W., Karki, S., Vafa, O., Peng, J. S., Hyun, L., Chan, C., Chung, H. S., Eivazi, A., Yoder,
S. C., Vielmetter, J., Carmichael, D. F., Hayes, R. J. and Dahiyat, B. I. (2006). "Engineered
antibody Fc variants with enhanced effector function." Proc Natl Acad Sci U S A 103(11):
4005-4010.
Lindquist, S. and Craig, E. A. (1988). "The heat-shock proteins." Annu Rev Genet 22: 631-677.
Lobo, E. D., Hansen, R. J. and Balthasar, J. P. (2004). "Antibody pharmacokinetics and
pharmacodynamics." J Pharm Sci 93(11): 2645-2668.
Locksley, R. M., Killeen, N. and Lenardo, M. J. (2001). "The TNF and TNF receptor superfamilies:
integrating mammalian biology." Cell 104(4): 487-501.
Loh, D. Y., Bothwell, A. L., White-Scharf, M. E., Imanishi-Kari, T. and Baltimore, D. (1983). "Molecular
basis of a mouse strain-specific anti-hapten response." Cell 33(1): 85-93.
Lonberg, N. (2005). "Human antibodies from transgenic animals." Nat Biotechnol 23(9): 1117-1125.
108
Kirstin A. Zettlitz
References
Lonberg, N., Taylor, L. D., Harding, F. A., Trounstine, M., Higgins, K. M., Schramm, S. R., Kuo, C. C.,
Mashayekh, R., Wymore, K., McCabe, J. G. and et al. (1994). "Antigen-specific human
antibodies from mice comprising four distinct genetic modifications." Nature 368(6474): 856859.
Low, N. M., Holliger, P. H. and Winter, G. (1996). "Mimicking somatic hypermutation: affinity
maturation of antibodies displayed on bacteriophage using a bacterial mutator strain." J Mol
Biol 260(3): 359-368.
Lowe, D. and Jermutus, L. (2004). "Combinatorial protein biochemistry for therapeutics and
proteomics." Curr Pharm Biotechnol 5(1): 17-27.
Luginbuhl, B., Kanyo, Z., Jones, R. M., Fletterick, R. J., Prusiner, S. B., Cohen, F. E., Williamson, R. A.,
Burton, D. R. and Pluckthun, A. (2006). "Directed evolution of an anti-prion protein scFv
fragment to an affinity of 1 pM and its structural interpretation." J Mol Biol 363(1): 75-97.
MacCallum, R. M., Martin, A. C. and Thornton, J. M. (1996). "Antibody-antigen interactions: contact
analysis and binding site topography." J Mol Biol 262(5): 732-745.
Makabe, K., Nakanishi, T., Tsumoto, K., Tanaka, Y., Kondo, H., Umetsu, M., Sone, Y., Asano, R. and
Kumagai, I. (2008). "Thermodynamic consequences of mutations in vernier zone residues of a
humanized anti-human epidermal growth factor receptor murine antibody, 528." J Biol Chem
283(2): 1156-1166.
Marchetti, L., Klein, M., Schlett, K., Pfizenmaier, K. and Eisel, U. L. (2004). "Tumor necrosis factor
(TNF)-mediated neuroprotection against glutamate-induced excitotoxicity is enhanced by Nmethyl-D-aspartate receptor activation. Essential role of a TNF receptor 2-mediated
phosphatidylinositol 3-kinase-dependent NF-kappa B pathway." J Biol Chem 279(31): 3286932881.
Marks, J. D., Griffiths, A. D., Malmqvist, M., Clackson, T. P., Bye, J. M. and Winter, G. (1992). "Bypassing immunization: building high affinity human antibodies by chain shuffling."
Biotechnology (N Y) 10(7): 779-783.
Martin, A. C. (2001). Protein Sequence and structural analysis of antibody variable domains, Springer
Heidelberg.
Martin, A. C. and Thornton, J. M. (1996). "Structural families in loops of homologous proteins:
automatic classification, modelling and application to antibodies." J Mol Biol 263(5): 800-815.
Mayer, M. P. and Bukau, B. (2005). "Hsp70 chaperones: cellular functions and molecular
mechanism." Cell Mol Life Sci 62(6): 670-684.
McCafferty, J., Griffiths, A. D., Winter, G. and Chiswell, D. J. (1990). "Phage antibodies: filamentous
phage displaying antibody variable domains." Nature 348(6301): 552-554.
Melder, R. J., Osborn, B. L., Riccobene, T., Kanakaraj, P., Wei, P., Chen, G., Stolow, D., Halpern, W. G.,
Migone, T. S., Wang, Q., Grzegorzewski, K. J. and Gallant, G. (2005). "Pharmacokinetics and in
vitro and in vivo anti-tumor response of an interleukin-2-human serum albumin fusion
protein in mice." Cancer Immunol Immunother 54(6): 535-547.
Mole, C. M., Bene, M. C., Montagne, P. M., Seilles, E. and Faure, G. C. (1994). "Light chains of
immunoglobulins in human secretions." Clin Chim Acta 224(2): 191-197.
Moosmayer, D., Dubel, S., Brocks, B., Watzka, H., Hampp, C., Scheurich, P., Little, M. and Pfizenmaier,
K. (1995). "A single-chain TNF receptor antagonist is an effective inhibitor of TNF mediated
cytotoxicity." Ther Immunol 2(1): 31-40.
Morrison, S. L., Johnson, M. J., Herzenberg, L. A. and Oi, V. T. (1984). "Chimeric human antibody
molecules: mouse antigen-binding domains with human constant region domains." Proc Natl
Acad Sci U S A 81(21): 6851-6855.
Moser, C., Schmidbauer, C., Gurtler, U., Gross, C., Gehrmann, M., Thonigs, G., Pfister, K. and
Multhoff, G. (2002). "Inhibition of tumor growth in mice with severe combined
immunodeficiency is mediated by heat shock protein 70 (Hsp70)-peptide-activated, CD94
positive natural killer cells." Cell Stress Chaperones 7(4): 365-373.
109
Kirstin A. Zettlitz
References
Muller, D., Karle, A., Meissburger, B., Hofig, I., Stork, R. and Kontermann, R. E. (2007). "Improved
pharmacokinetics of recombinant bispecific antibody molecules by fusion to human serum
albumin." J Biol Chem 282(17): 12650-12660.
Muller, D., Trunk, G., Sichelstiel, A., Zettlitz, K. A., Quintanilla, M. and Kontermann, R. E. (2008).
"Murine endoglin-specific single-chain Fv fragments for the analysis of vascular targeting
strategies in mice." J Immunol Methods 339(1): 90-98.
Muller, N., Schneider, B., Pfizenmaier, K. and Wajant, H. (2010). "Superior serum half life of albumin
tagged TNF ligands." Biochem Biophys Res Commun 396(4): 793-799.
Multhoff, G. (2002). "Activation of natural killer cells by heat shock protein 70." Int J Hyperthermia
18(6): 576-585.
Multhoff, G. (2007). "Heat shock protein 70 (Hsp70): membrane location, export and immunological
relevance." Methods 43(3): 229-237.
Multhoff, G. (2009). "Activation of natural killer cells by heat shock protein 70. 2002." Int J
Hyperthermia 25(3): 169-175.
Multhoff, G., Botzler, C., Jennen, L., Schmidt, J., Ellwart, J. and Issels, R. (1997). "Heat shock protein
72 on tumor cells: a recognition structure for natural killer cells." J Immunol 158(9): 43414350.
Multhoff, G., Botzler, C., Wiesnet, M., Muller, E., Meier, T., Wilmanns, W. and Issels, R. D. (1995). "A
stress-inducible 72-kDa heat-shock protein (HSP72) is expressed on the surface of human
tumor cells, but not on normal cells." Int J Cancer 61(2): 272-279.
Multhoff, G., Pfister, K., Botzler, C., Jordan, A., Scholz, R., Schmetzer, H., Burgstahler, R. and
Hiddemann, W. (2000). "Adoptive transfer of human natural killer cells in mice with severe
combined immunodeficiency inhibits growth of Hsp70-expressing tumors." Int J Cancer 88(5):
791-797.
Multhoff, G., Pfister, K., Gehrmann, M., Hantschel, M., Gross, C., Hafner, M. and Hiddemann, W.
(2001). "A 14-mer Hsp70 peptide stimulates natural killer (NK) cell activity." Cell Stress
Chaperones 6(4): 337-344.
Musolino, A., Naldi, N., Bortesi, B., Pezzuolo, D., Capelletti, M., Missale, G., Laccabue, D., Zerbini, A.,
Camisa, R., Bisagni, G., Neri, T. M. and Ardizzoni, A. (2008). "Immunoglobulin G fragment C
receptor polymorphisms and clinical efficacy of trastuzumab-based therapy in patients with
HER-2/neu-positive metastatic breast cancer." J Clin Oncol 26(11): 1789-1796.
Myszka, D. G. (1997). "Kinetic analysis of macromolecular interactions using surface plasmon
resonance biosensors." Curr Opin Biotechnol 8(1): 50-57.
Myszka, D. G. and Morton, T. A. (1998). "CLAMP: a biosensor kinetic data analysis program." Trends
Biochem Sci 23(4): 149-150.
Natsume, A., In, M., Takamura, H., Nakagawa, T., Shimizu, Y., Kitajima, K., Wakitani, M., Ohta, S.,
Satoh, M., Shitara, K. and Niwa, R. (2008). "Engineered antibodies of IgG1/IgG3 mixed isotype
with enhanced cytotoxic activities." Cancer Res 68(10): 3863-3872.
Natsume, A., Niwa, R. and Satoh, M. (2009). "Improving effector functions of antibodies for cancer
treatment: Enhancing ADCC and CDC." Drug Des Devel Ther 3: 7-16.
Nielsen, U. B., Adams, G. P., Weiner, L. M. and Marks, J. D. (2000). "Targeting of bivalent anti-ErbB2
diabody antibody fragments to tumor cells is independent of the intrinsic antibody affinity."
Cancer Res 60(22): 6434-6440.
Nimmerjahn, F. and Ravetch, J. V. (2006). "Fcgamma receptors: old friends and new family
members." Immunity 24(1): 19-28.
Novotny, J., Bruccoleri, R. E. and Saul, F. A. (1989). "On the attribution of binding energy in antigenantibody complexes McPC 603, D1.3, and HyHEL-5." Biochemistry 28(11): 4735-4749.
Novotny, J., Handschumacher, M., Haber, E., Bruccoleri, R. E., Carlson, W. B., Fanning, D. W., Smith, J.
A. and Rose, G. D. (1986). "Antigenic determinants in proteins coincide with surface regions
accessible to large probes (antibody domains)." Proc Natl Acad Sci U S A 83(2): 226-230.
110
Kirstin A. Zettlitz
References
Ober, R. J., Radu, C. G., Ghetie, V. and Ward, E. S. (2001). "Differences in promiscuity for antibodyFcRn interactions across species: implications for therapeutic antibodies." Int Immunol
13(12): 1551-1559.
Oflazoglu, E. and Audoly, L. P. (2010). "Evolution of anti-CD20 monoclonal antibody therapeutics in
oncology." MAbs 2(1): 14-19.
Olleros, M. L., Vesin, D., Lambou, A. F., Janssens, J. P., Ryffel, B., Rose, S., Fremond, C., Quesniaux, V.
F., Szymkowski, D. E. and Garcia, I. (2009). "Dominant-negative tumor necrosis factor
protects from Mycobacterium bovis Bacillus Calmette Guerin (BCG) and endotoxin-induced
liver injury without compromising host immunity to BCG and Mycobacterium tuberculosis." J
Infect Dis 199(7): 1053-1063.
Ow, D. S., Lim, D. Y., Nissom, P. M., Camattari, A. and Wong, V. V. (2010). "Co-expression of Skp and
FkpA chaperones improves cell viability and alters the global expression of stress response
genes during scFvD1.3 production." Microb Cell Fact 9: 22.
Padlan, E. A. (1991). "A possible procedure for reducing the immunogenicity of antibody variable
domains while preserving their ligand-binding properties." Mol Immunol 28(4-5): 489-498.
Perelson, A. S. and Oster, G. F. (1979). "Theoretical studies of clonal selection: minimal antibody
repertoire size and reliability of self-non-self discrimination." J Theor Biol 81(4): 645-670.
Pfister, K., Radons, J., Busch, R., Tidball, J. G., Pfeifer, M., Freitag, L., Feldmann, H. J., Milani, V., Issels,
R. and Multhoff, G. (2007). "Patient survival by Hsp70 membrane phenotype: association
with different routes of metastasis." Cancer 110(4): 926-935.
Pope, R. M. (2002). "Apoptosis as a therapeutic tool in rheumatoid arthritis." Nat Rev Immunol 2(7):
527-535.
Popkov, M., Jendreyko, N., Gonzalez-Sapienza, G., Mage, R. G., Rader, C. and Barbas, C. F., 3rd (2004).
"Human/mouse cross-reactive anti-VEGF receptor 2 recombinant antibodies selected from
an immune b9 allotype rabbit antibody library." J Immunol Methods 288(1-2): 149-164.
Presta, L. G. (2002). "Engineering antibodies for therapy." Curr Pharm Biotechnol 3(3): 237-256.
Presta, L. G. (2006). "Engineering of therapeutic antibodies to minimize immunogenicity and optimize
function." Adv Drug Deliv Rev 58(5-6): 640-656.
Presta, L. G. (2008). "Molecular engineering and design of therapeutic antibodies." Curr Opin
Immunol 20(4): 460-470.
Qiao, S. W., Kobayashi, K., Johansen, F. E., Sollid, L. M., Andersen, J. T., Milford, E., Roopenian, D. C.,
Lencer, W. I. and Blumberg, R. S. (2008). "Dependence of antibody-mediated presentation of
antigen on FcRn." Proc Natl Acad Sci U S A 105(27): 9337-9342.
Racila, E., Link, B. K., Weng, W. K., Witzig, T. E., Ansell, S., Maurer, M. J., Huang, J., Dahle, C., Halwani,
A., Levy, R. and Weiner, G. J. (2008). "A polymorphism in the complement component C1qA
correlates with prolonged response following rituximab therapy of follicular lymphoma." Clin
Cancer Res 14(20): 6697-6703.
Razai, A., Garcia-Rodriguez, C., Lou, J., Geren, I. N., Forsyth, C. M., Robles, Y., Tsai, R., Smith, T. J.,
Smith, L. A., Siegel, R. W., Feldhaus, M. and Marks, J. D. (2005). "Molecular evolution of
antibody affinity for sensitive detection of botulinum neurotoxin type A." J Mol Biol 351(1):
158-169.
Reichert, J. M. (2010). "Antibodies to watch in 2010." MAbs 2(1): 84-100.
Reichert, J. M., Rosensweig, C. J., Faden, L. B. and Dewitz, M. C. (2005). "Monoclonal antibody
successes in the clinic." Nat Biotechnol 23(9): 1073-1078.
Roopenian, D. C. and Akilesh, S. (2007). "FcRn: the neonatal Fc receptor comes of age." Nat Rev
Immunol 7(9): 715-725.
Saldanha, J. W., Martin, A. C. and Leger, O. J. (1999). "A single backmutation in the human kIV
framework of a previously unsuccessfully humanized antibody restores the binding activity
and increases the secretion in cos cells." Mol Immunol 36(11-12): 709-719.
Salfeld, J. G. (2007). "Isotype selection in antibody engineering." Nat Biotechnol 25(12): 1369-1372.
111
Kirstin A. Zettlitz
References
Schier, R., Balint, R. F., McCall, A., Apell, G., Larrick, J. W. and Marks, J. D. (1996a). "Identification of
functional and structural amino-acid residues by parsimonious mutagenesis." Gene 169(2):
147-155.
Schier, R., Bye, J., Apell, G., McCall, A., Adams, G. P., Malmqvist, M., Weiner, L. M. and Marks, J. D.
(1996b). "Isolation of high-affinity monomeric human anti-c-erbB-2 single chain Fv using
affinity-driven selection." J Mol Biol 255(1): 28-43.
Schier, R., McCall, A., Adams, G. P., Marshall, K. W., Merritt, H., Yim, M., Crawford, R. S., Weiner, L.
M., Marks, C. and Marks, J. D. (1996c). "Isolation of picomolar affinity anti-c-erbB-2 singlechain Fv by molecular evolution of the complementarity determining regions in the center of
the antibody binding site." J Mol Biol 263(4): 551-567.
Schiff, M. H., Burmester, G. R., Kent, J. D., Pangan, A. L., Kupper, H., Fitzpatrick, S. B. and Donovan, C.
(2006). "Safety analyses of adalimumab (HUMIRA) in global clinical trials and US
postmarketing surveillance of patients with rheumatoid arthritis." Ann Rheum Dis 65(7): 889894.
Schlapschy, M., Fogarasi, M., Gruber, H., Gresch, O., Schafer, C., Aguib, Y. and Skerra, A. (2009).
"Functional humanization of an anti-CD16 Fab fragment: obstacles of switching from murine
{lambda} to human {lambda} or {kappa} light chains." Protein Eng Des Sel 22(3): 175-188.
Schlapschy, M., Gruber, H., Gresch, O., Schafer, C., Renner, C., Pfreundschuh, M. and Skerra, A.
(2004). "Functional humanization of an anti-CD30 Fab fragment for the immunotherapy of
Hodgkin's lymphoma using an in vitro evolution approach." Protein Eng Des Sel 17(12): 847860.
Seitter, J. (2010). "Immunaffine und immunstimulatorische Strategien zur Tumortherapie." PhD
Thesis. Universität Konstanz
Sherman, M. and Multhoff, G. (2007). "Heat shock proteins in cancer." Ann N Y Acad Sci 1113: 192201.
Shibata, H., Yoshioka, Y., Abe, Y., Ohkawa, A., Nomura, T., Minowa, K., Mukai, Y., Nakagawa, S.,
Taniai, M., Ohta, T., Kamada, H., Tsunoda, S. and Tsutsumi, Y. (2009). "The treatment of
established murine collagen-induced arthritis with a TNFR1-selective antagonistic mutant
TNF." Biomaterials 30(34): 6638-6647.
Shibata, H., Yoshioka, Y., Ohkawa, A., Abe, Y., Nomura, T., Mukai, Y., Nakagawa, S., Taniai, M., Ohta,
T., Mayumi, T., Kamada, H., Tsunoda, S. and Tsutsumi, Y. (2008a). "The therapeutic effect of
TNFR1-selective antagonistic mutant TNF-alpha in murine hepatitis models." Cytokine 44(2):
229-233.
Shibata, H., Yoshioka, Y., Ohkawa, A., Minowa, K., Mukai, Y., Abe, Y., Taniai, M., Nomura, T.,
Kayamuro, H., Nabeshi, H., Sugita, T., Imai, S., Nagano, K., Yoshikawa, T., Fujita, T., Nakagawa,
S., Yamamoto, A., Ohta, T., Hayakawa, T., Mayumi, T., Vandenabeele, P., Aggarwal, B. B.,
Nakamura, T., Yamagata, Y., Tsunoda, S., Kamada, H. and Tsutsumi, Y. (2008b). "Creation and
X-ray structure analysis of the tumor necrosis factor receptor-1-selective mutant of a tumor
necrosis factor-alpha antagonist." J Biol Chem 283(2): 998-1007.
Shields, R. L., Namenuk, A. K., Hong, K., Meng, Y. G., Rae, J., Briggs, J., Xie, D., Lai, J., Stadlen, A., Li, B.,
Fox, J. A. and Presta, L. G. (2001). "High resolution mapping of the binding site on human
IgG1 for Fc gamma RI, Fc gamma RII, Fc gamma RIII, and FcRn and design of IgG1 variants
with improved binding to the Fc gamma R." J Biol Chem 276(9): 6591-6604.
Siegel, R. M., Frederiksen, J. K., Zacharias, D. A., Chan, F. K., Johnson, M., Lynch, D., Tsien, R. Y. and
Lenardo, M. J. (2000). "Fas preassociation required for apoptosis signaling and dominant
inhibition by pathogenic mutations." Science 288(5475): 2354-2357.
Smith, B. J., Popplewell, A., Athwal, D., Chapman, A. P., Heywood, S., West, S. M., Carrington, B.,
Nesbitt, A., Lawson, A. D., Antoniw, P., Eddelston, A. and Suitters, A. (2001). "Prolonged in
vivo residence times of antibody fragments associated with albumin." Bioconjug Chem 12(5):
750-756.
112
Kirstin A. Zettlitz
References
Smith, K. G. and Clatworthy, M. R. (2010). "FcgammaRIIB in autoimmunity and infection: evolutionary
and therapeutic implications." Nat Rev Immunol 10(5): 328-343.
Spector, N. L. and Blackwell, K. L. (2009). "Understanding the mechanisms behind trastuzumab
therapy for human epidermal growth factor receptor 2-positive breast cancer." J Clin Oncol
27(34): 5838-5847.
Stangl, S., Gehrmann, M., Dressel, R., Alves, F., Dullin, C., Themelis, G., Ntziachristos, V., Staeblein, E.,
Walch, A., Winkelmann, I. and Multhoff, G. (2010). "In vivo imaging of CT26 mouse tumors by
using cmHsp70.1 monoclonal antibody." J Cell Mol Med.
Steigerwald, J., Raum, T., Pflanz, S., Cierpka, R., Mangold, S., Rau, D., Hoffmann, P., Kvesic, M., Zube,
C., Linnerbauer, S., Lumsden, J., Sriskandarajah, M., Kufer, P., Baeuerle, P. A. and Volkland, J.
(2009). "Human IgG1 antibodies antagonizing activating receptor NKG2D on natural killer
cells." MAbs 1(2): 115-127.
Steiner, K., Graf, M., Hecht, K., Reif, S., Rossbacher, L., Pfister, K., Kolb, H. J., Schmetzer, H. M. and
Multhoff, G. (2006). "High HSP70-membrane expression on leukemic cells from patients with
acute myeloid leukemia is associated with a worse prognosis." Leukemia 20(11): 2076-2079.
Stork, R., Zettlitz, K. A., Muller, D., Rether, M., Hanisch, F. G. and Kontermann, R. E. (2008). "Nglycosylation as novel strategy to improve pharmacokinetic properties of bispecific singlechain diabodies." J Biol Chem 283(12): 7804-7812.
Strohl, W. R. (2009). "Optimization of Fc-mediated effector functions of monoclonal antibodies." Curr
Opin Biotechnol 20(6): 685-691.
Szymkowski, D. E. (2005). "Creating the next generation of protein therapeutics through rational drug
design." Curr Opin Drug Discov Devel 8(5): 590-600.
t Hart, B. A., Amor, S. and Jonker, M. (2004). "Evaluating the validity of animal models for research
into therapies for immune-based disorders." Drug Discov Today 9(12): 517-524.
Tatusova, T. A. and Madden, T. L. (1999). "BLAST 2 Sequences, a new tool for comparing protein and
nucleotide sequences." FEMS Microbiol Lett 174(2): 247-250.
Terato, K., Arai, H., Shimozuru, Y., Fukuda, T., Tanaka, H., Watanabe, H., Nagai, Y., Fujimoto, K.,
Okubo, F., Cho, F. and et al. (1989). "Sex-linked differences in susceptibility of cynomolgus
monkeys to type II collagen-induced arthritis. Evidence that epitope-specific immune
suppression is involved in the regulation of type II collagen autoantibody formation."
Arthritis Rheum 32(6): 748-758.
Thoma, B., Grell, M., Pfizenmaier, K. and Scheurich, P. (1990). "Identification of a 60-kD tumor
necrosis factor (TNF) receptor as the major signal transducing component in TNF responses."
J Exp Med 172(4): 1019-1023.
Tomlinson, I. M., Walter, G., Jones, P. T., Dear, P. H., Sonnhammer, E. L. and Winter, G. (1996). "The
imprint of somatic hypermutation on the repertoire of human germline V genes." J Mol Biol
256(5): 813-817.
Tracey, D., Klareskog, L., Sasso, E. H., Salfeld, J. G. and Tak, P. P. (2008). "Tumor necrosis factor
antagonist mechanisms of action: a comprehensive review." Pharmacol Ther 117(2): 244279.
Valjakka, J., Hemminki, A., Niemi, S., Soderlund, H., Takkinen, K. and Rouvinen, J. (2002). "Crystal
structure of an in vitro affinity- and specificity-matured anti-testosterone Fab in complex
with testosterone. Improved affinity results from small structural changes within the variable
domains." J Biol Chem 277(46): 44021-44027.
Vega, V. L., Rodriguez-Silva, M., Frey, T., Gehrmann, M., Diaz, J. C., Steinem, C., Multhoff, G., Arispe,
N. and De Maio, A. (2008). "Hsp70 translocates into the plasma membrane after stress and is
released into the extracellular environment in a membrane-associated form that activates
macrophages." J Immunol 180(6): 4299-4307.
Verhoeyen, M., Milstein, C. and Winter, G. (1988). "Reshaping human antibodies: grafting an
antilysozyme activity." Science 239(4847): 1534-1536.
113
Kirstin A. Zettlitz
References
Vierboom, M. P., Jonker, M., Tak, P. P. and t Hart, B. A. (2007). "Preclinical models of arthritic disease
in non-human primates." Drug Discov Today 12(7-8): 327-335.
Vultaggio, A., Matucci, A., Nencini, F., Pratesi, S., Parronchi, P., Rossi, O., Romagnani, S. and Maggi, E.
(2009). "Anti-infliximab IgE and non-IgE antibodies and induction of infusion-related severe
anaphylactic reactions." Allergy 65(5): 657-661.
Wajant, H., Pfizenmaier, K. and Scheurich, P. (2003). "Tumor necrosis factor signaling." Cell Death
Differ 10(1): 45-65.
Wallach, D. and Kovalenko, A. (2009). "12th international TNF conference: the good, the bad and the
scientists." Cytokine Growth Factor Rev 20(4): 259-269.
Wallis, R. S. (2008). "Tumour necrosis factor antagonists: structure, function, and tuberculosis risks."
Lancet Infect Dis 8(10): 601-611.
Wark, K. L. and Hudson, P. J. (2006). "Latest technologies for the enhancement of antibody affinity."
Adv Drug Deliv Rev 58(5-6): 657-670.
Weiner, L. M., Surana, R. and Wang, S. (2010). "Monoclonal antibodies: versatile platforms for cancer
immunotherapy." Nat Rev Immunol 10(5): 317-327.
Wells, J. A. (1990). "Additivity of mutational effects in proteins." Biochemistry 29(37): 8509-8517.
Wilson, N. S., Dixit, V. and Ashkenazi, A. (2009). "Death receptor signal transducers: nodes of
coordination in immune signaling networks." Nat Immunol 10(4): 348-355.
Worn, A., Auf der Maur, A., Escher, D., Honegger, A., Barberis, A. and Pluckthun, A. (2000).
"Correlation between in vitro stability and in vivo performance of anti-GCN4 intrabodies as
cytoplasmic inhibitors." J Biol Chem 275(4): 2795-2803.
Wright, A. and Morrison, S. L. (1993). "Antibody variable region glycosylation: biochemical and
clinical effects." Springer Semin Immunopathol 15(2-3): 259-273.
Wu, H., Pfarr, D. S., Tang, Y., An, L. L., Patel, N. K., Watkins, J. D., Huse, W. D., Kiener, P. A. and Young,
J. F. (2005). "Ultra-potent antibodies against respiratory syncytial virus: effects of binding
kinetics and binding valence on viral neutralization." J Mol Biol 350(1): 126-144.
Yang, W. P., Green, K., Pinz-Sweeney, S., Briones, A. T., Burton, D. R. and Barbas, C. F., 3rd (1995).
"CDR walking mutagenesis for the affinity maturation of a potent human anti-HIV-1 antibody
into the picomolar range." J Mol Biol 254(3): 392-403.
Yau, K. Y., Dubuc, G., Li, S., Hirama, T., Mackenzie, C. R., Jermutus, L., Hall, J. C. and Tanha, J. (2005).
"Affinity maturation of a V(H)H by mutational hotspot randomization." J Immunol Methods
297(1-2): 213-224.
Zalevsky, J., Chamberlain, A. K., Horton, H. M., Karki, S., Leung, I. W., Sproule, T. J., Lazar, G. A.,
Roopenian, D. C. and Desjarlais, J. R. (2010). "Enhanced antibody half-life improves in vivo
activity." Nat Biotechnol 28(2): 157-159.
Zalevsky, J., Secher, T., Ezhevsky, S. A., Janot, L., Steed, P. M., O'Brien, C., Eivazi, A., Kung, J., Nguyen,
D. H., Doberstein, S. K., Erard, F., Ryffel, B. and Szymkowski, D. E. (2007). "Dominant-negative
inhibitors of soluble TNF attenuate experimental arthritis without suppressing innate
immunity to infection." J Immunol 179(3): 1872-1883.
Zeman, K., Kantorski, J., Paleolog, E. M., Feldmann, M. and Tchorzewski, H. (1996). "The role of
receptors for tumour necrosis factor-alpha in the induction of human polymorphonuclear
neutrophil chemiluminescence." Immunol Lett 53(1): 45-50.
Zettlitz, K. A. (2007). "Recombinant Hsp70-specific Antibodies for Cancer Therapy." Diploma Thesis.
Zettlitz, K. A., Seitter, J., Muller, D. and Kontermann, R. E. (2010). "Humanization of a Mouse
Monoclonal Antibody Directed Against a Cell Surface-Exposed Epitope of MembraneAssociated Heat Shock Protein 70 (Hsp70)." Mol Biotechnol.
114
Kirstin A. Zettlitz
Sequences
6 Sequences
6.1 human Hsp70
1
81
161
241
321
401
481
561
641
721
801
881
961
1041
1121
1201
1281
1361
1441
1521
1601
1681
atggccaaag ccgcggcgat cggcatcgac ctgggcacca cctactcctg cgtgggggtg ttccaacacg gcaaggtgga
>>...................................ATPase domain....................................>
m a k
a a a
i g i d
l g t
t y s
c v g v
f q h
g k v
gatcatcgcc aacgaccagg gcaaccgcac cacccccagc tacgtggcct tcacggacac cgagcggctc atcggggatg
>....................................ATPase domain....................................>
e i i a
n d q
g n r
t t p s
y v a
f t d
t e r l
i g d
cggccaagaa ccaggtggcg ctgaacccgc agaacaccgt gtttgacgcg aagcgcctga ttggccgcaa gttcggcgac
>....................................ATPase domain....................................>
a a k
n q v a
l n p
q n t
v f d a
k r l
i g r
k f g d
ccggtggtgc agtcggacat gaagcactgg cctttccagg tgatcaacga cggagacaag cccaaggtgc aggtgagcta
>....................................ATPase domain....................................>
p v v
q s d
m k h w
p f q
v i n
d g d k
p k v
q v s
caagggggag accaaggcat tctaccccga ggagatctcg tccatggtgc tgaccaagat gaaggagatc gccgaggcgt
>....................................ATPase domain....................................>
y k g e
t k a
f y p
e e i s
s m v
l t k
m k e i
a e a
acctgggcta cccggtgacc aacgcggtga tcaccgtgcc ggcctacttc aacgactcgc agcgccaggc caccaaggat
>....................................ATPase domain....................................>
y l g
y p v t
n a v
i t v
p a y f
n d s
q r q
a t k d
gcgggtgtga tcgcggggct caacgtgctg cggatcatca acgagcccac ggccgccgcc atcgcctacg gcctggacag
>....................................ATPase domain....................................>
a g v
i a g
l n v l
r i i
n e p
t a a a
i a y
g l d
aacgggcaag ggggagcgca acgtgctcat ctttgacctg ggcgggggca ccttcgacgt gtccatcctg acgatcgacg
>....................................ATPase domain....................................>
r t g k
g e r
n v l
i f d l
g g g
t f d
v s i l
t i d
acggcatctt cgaggtgaag gccacggccg gggacaccca cctgggtggg gaggactttg acaacaggct ggtgaaccac
>....................................ATPase domain....................................>
d g i
f e v k
a t a
g d t
h l g g
e d f
d n r
l v n h
ttcgtggagg agttcaagag aaaacacaag aaggacatca gccagaacaa gcgagccgtg aggcggctgc gcaccgcctg
>....................................ATPase domain....................................>
f v e
e f k
r k h k
k d i
s q n
k r a v
r r l
r t a
cgagagggcc aagaggaccc tgtcgtccag cacccaggcc agcctggaga tcgactccct gtttgagggc atcgacttct
>....................................ATPase domain....................................>
c e r a
k r t
l s s
s t q a
s l e
i d s
l f e g
i d f
acacgtccat caccagggcg aggttcgagg agctgtgctc cgacctgttc cgaagcaccc tggagcccgt ggagaaggct
>....................................ATPase domain....................................>
y t s
i t r a
r f e
e l c
s d l f
r s t
l e p
v e k a
ctgcgcgacg ccaagctgga caaggcccag attcacgacc tggtcctggt cgggggctcc acccgcatcc ccaaggtgca
>....................................ATPase domain....................................>
l r d
a k l
d k a q
i h d
l v l
v g g s
t r i
p k v
gaagctgctg caggacttct tcaacgggcg cgacctgaac aagagcatca accccgacga ggctgtggcc tacggggcgg
>....................................ATPase domain....................................>
q k l l
q d f
f n g
r d l n
k s i
n p d
e a v a
y g a
cggtgcaggc ggccatcctg atgggggaca agtccgagaa cgtgcaggac ctgctgctgc tggacgtggc tcccctgtcg
>......ATPase domain......>>>>...............Substrate binding domain.................>
a v q
a a i l
m g d
k s e
n v q d
l l l
l d v
a p l s
ctggggctgg agacggccgg aggcgtgatg actgccctga tcaagcgcaa ctccaccatc cccaccaagc agacgcagat
>..............................Substrate binding domain...............................>
l g l
e t a
g g v m
t a l
i k r
n s t i
p t k
q t q
cttcaccacc tactccgaca accaacccgg ggtgctgatc caggtgtacg agggcgagag ggccatgacg aaagacaaca
i f t t
y s d
n q p
g v l i
q v y
e g e
r a m t
k d n
atctgttggg gcgcttcgag ctgagcggca tccctccggc ccccaggggc gtgccccaga tcgaggtgac cttcgacatc
n l l
g r f e
l s g
i p p
a p r g
v p q
i e v
t f d i
gatgccaacg gcatcctgaa cgtcacggcc acggacaaga gcaccggcaa ggccaacaag atcaccatca ccaacgacaa
d a n
g i l
n v t a
t d k
s t g
k a n k
i t i
t n d
gggccgcctg agcaaggagg agatcgagcg catggtgcag gaggcggaga agtacaaagc ggaggacgag gtgcagcgcg
k g r l
s k e
e i e
r m v q
e a e
k y k
a e d e
v q r
agagggtgtc agccaagaac gccctggagt cctacgcctt caacatgaag agcgccgtgg aggatgaggg gctcaagggc
>.......................>>>>....................C-terminal domain.....................>
e r v
s a k n
a l e
s y a
f n m k
s a v
e d e
g l k g
aagatcagcg aggccgacaa gaagaaggtg ctggacaagt gtcaagaggt catctcgtgg ctggacgcca acaccttggc
>..................................C-terminal domain..................................>
k i s
e a d
k k k v
l d k
c q e
v i s w
l d a
n t l
115
Kirstin A. Zettlitz
1761
1841
1921
Sequences
cgagaaggac gagtttgagc acaagaggaa ggagctggag caggtgtgta accccatcat cagcggactg taccagggtg
a e k d
e f e
h k r
k e l e
q v c
n p i
i s g l
y q g
ccggtggtcc cgggcctggg ggcttcgggg ctcagggtcc caagggaggg tctgggtcag gccccaccat tgaggaggta
a g g
p g p g
g f g
a q g
p k g g
s g s
g p t
i e e v
gattag
>>> C-terminal domain
d *
6.2 human TNFR1-Fc
AgeI
-+---1 atggagacag acacactcct gctatgggta ctgctgctct gggttccagg ttccaccggt ctggtccctc acctaggcga
Igk-Leader'
>>.N-terminal peptide...>
g
l v p
h l g
81 tcgggagaag agagatagtg tgtgtcccca aggaaaatat atccaccctc aaaataattc gatttgctgt acaaagtgcc
...........>>>>.........................CDR1.A1.......................>>>>...CRD1.B2....>
d r e k
r d s
v c p
q g k y
i h p
q n n
s i c c
t k c
161 acaaaggaac ctacttgtat aatgactgtc caggcccggg gcaggatacg gactgcaggg agtgtgagag cggctccttc
>..............................B2.............................>>>>.......CRD2.........>
h k g
t y l y
n d c
p g p
g q d t
d c r
e c e
s g s f
241 accgcttcag aaaaccacct cagacactgc ctcagctgct ccaaatgccg aaaggaaatg ggtcaggtgg agatctcttc
>........................................CRD2.........................................>
t a s
e n h
l r h c
l s c
s k c
r k e m
g q v
e i s
321 ttgcacagtg gaccgggaca ccgtgtgtgg ctgtaggaag aaccagtacc ggcattattg gagtgaaaac cttttccagt
>..............CRD2.............>>>>......................CRD3........................>
s c t v
d r d
t v c
g c r k
n q y
r h y
w s e n
l f q
401 gcttcaattg cagcctctgc ctcaatggga ccgtgcacct ctcctgccag gagaaacaga ataccgtgtg cacctgccat
>.....................................CRD3.....................................>>>>...>
c f n
c s l c
l n g
t v h
l s c q
e k q
n t v
c t c h
481 gcaggtttct ttctaagaga aaacgagtgt gtctcctgta gtaactgtaa gaaaagcctg gagtgcacga agttgtgcct
>........................................CRD4.........................................>
a g f
f l r
e n e c
v s c
s n c
k k s l
e c t
k l c
NotI
--+-----561 accccagatt gagaatgtta agggcactga ggactcaggt accacagcgg ccgcagacaa aactcacaca tgcccaccgt
>.>>>>...................stem...................>>
>>.........hinge..........>
l p q i
e n v
k g t
e d s g
t t
d
k t h t
c p p
641 gcccagcacc tgaactcctg gggggaccgt cagtcttcct cttcccccca aaacccaagg acaccctcat gatctcccgg
>..>>>>.....................................CH2.......................................>
c p a
p e l l
g g p
s v f
l f p p
k p k
d t l
m i s r
721 acccctgagg tcacatgcgt ggtggtggac gtgagccacg aagaccctga ggtcaagttc aactggtacg tggacggcgt
>.........................................CH2.........................................>
t p e
v t c
v v v d
v s h
e d p
e v k f
n w y
v d g
801 ggaggtgcat aatgccaaga caaagccgcg ggaggagcag tacaacagca cgtaccgggt ggtcagcgtc ctcaccgtcc
>.........................................CH2.........................................>
v e v h
n a k
t k p
r e e q
y n s
t y r
v v s v
l t v
881 tgcaccagga ctggctgaat ggcaaggagt acaagtgcaa ggtctccaac aaagccctcc cagcccccat cgagaaaacc
>.........................................CH2.........................................>
l h q
d w l n
g k e
y k c
k v s n
k a l
p a p
i e k t
961 atctccaaag ccaaagggca gccccgagaa ccacaggtgt acaccctgcc cccatcccgg gaggagatga ccaagaacca
>.....CH2.....>>>>................................CH3.................................>
i s k
a k g
q p r e
p q v
y t l
p p s r
e e m
t k n
1041 ggtcagcctg acctgcctgg tcaaaggctt ctatcccagc gacatcgccg tggagtggga gagcaatggg cagccggaga
>.........................................CH3.........................................>
q v s l
t c l
v k g
f y p s
d i a
v e w
e s n g
q p e
1121 acaactacaa gaccacgcct cccgtgctgg actccgacgg ctccttcttc ctctatagca agctcaccgt ggacaagagc
>.........................................CH3.........................................>
n n y
k t t p
p v l
d s d
g s f f
l y s
k l t
v d k s
1201
1281
aggtggcagc aggggaacgt cttctcatgc tccgtgatgc atgaggctct gcacaaccac tacacgcaga agagcctctc
>.........................................CH3.........................................>
r w q
q g n
v f s c
s v m
h e a
l h n h
y t q
k s l
EcoRI
--+---cctgtctccg ggtaaataag aattc
s l s p
g k
116
Kirstin A. Zettlitz
Sequences
6.3 mouse TNFR1
1
81
161
241
321
401
481
561
atggagacag acacactcct gctatgggta ctgctgctct gggttccagg ttccaccggt ctagtccctt ctcttggcga
Igk-Leader'
N-terminal peptide >>..................>
l v p
s l g
tcgggagaag agggatagct tgtgtcccca aggaaagtat gtccattcta agaacaattc catctgctgt acaaagtgcc
>...........>>>>.........................CRD1.A1..........................>>>>..B2...>
d r e k
r d s
l c p
q g k y
v h s
k n n
s i c c
t k c
acaaaggaac ctacttggtg agtgactgtc cgagcccagg gcgggataca gtctgcaggg agtgtgaaaa gggcaccttt
>...........................CRD1.B2...........................>>>>.......CRD2.........>
h k g
t y l v
s d c
p s p
g r d t
v c r
e c e
k g t f
acggcttccc agaattacct caggcagtgt ctcagttgca agacatgtcg gaaagaaatg tcccaggtgg agatctctcc
>........................................CRD2.........................................>
t a s
q n y
l r q c
l s c
k t c
r k e m
s q v
e i s
ttgccaagct gacaaggaca ccgtgtgtgg ctgtaaggag aaccagttcc aacgctacct gagtgagaca cacttccagt
>..............CRD2.............>>>>......................CRD3........................>
p c q a
d k d
t v c
g c k e
n q f
q r y
l s e t
h f q
gcgtggactg tagcccctgc ttcaacggca ccgtgacaat cccctgtaag gagactcaga ataccgtgtg taactgccat
>.....................................CRD3.....................................>>>>...>
c v d
c s p c
f n g
t v t
i p c k
e t q
n t v
c n c h
gcagggttct ttctgagaga aagtgagtgc gtcccttgca gccactgcaa gaaaaatgag gagtgtatga agttgtgcct
>........................................CRD4.........................................>
a g f
f l r
e s e c
v p c
s h c
k k n e
e c m
k l c
acctcctccg cttgcaaatg tcacaaaccc ccaggactca ggtaccgcgg ccgcagacaa aactcacaca tgcccaccgt
>.>>>>.................stem..................>>
>>.........hinge..........>
l p p p
l a n
v t n
p q d s
d
k t h t
c p p
6.4 rhesus TNFR1
1
81
161
241
321
401
481
561
atggagacag acacactcct gctatgggta ctgctgctct gggttccagg ttccaccggt ctggtgcccc ccctgcggga
Igk-Leader
N-terminal peptide >>..................>
l v p
p l r
ccgggagaag agggacagcg tgtgccccca gggcaagtac atccaccccc agaacaactc cgtgtgctgt acaaagtgcc
>...........>>>>................................CRD1..................................>
d r e k
r d s
v c p
q g k y
i h p
q n n
s v c c
t k c
acaagggcac ctacctgtat aacgactgcc ccggacccgg ccaggacacc gactgcaggg aatgcgagag cggcagcttc
>.............................CRD1............................>>>>.......CRD2.........>
h k g
t y l y
n d c
p g p
g q d t
d c r
e c e
s g s f
accgccagcg agaaccacct gcggcactgc ctgtcctgct ccaagtgccg gaaagaaatg ggccaggtgg agatcagcag
>........................................CRD2.........................................>
t a s
e n h
l r h c
l s c
s k c
r k e m
g q v
e i s
ctgcaccgtg gaccgggaca ccgtgtgtgg ctgccggaag aaccagtaca gatactattg gagcgagaac ctgttccagt
>............CRD2...........>> >>........................CRD3..........................>
s c t v
d r d
t v c
g c r k
n q y
r y y
w s e n
l f q
gcttcaactg ctccctgtgc ctgaacggca ccgtgcacct gagctgccaa gaaaagcaga ataccgtctg cacctgccac
>.....................................CRD3.....................................>>>>...>
c f n
c s l c
l n g
t v h
l s c q
e k q
n t v
c t c h
gccggctttt ttctgcggga gaacgagtgc gtgtcctgtt ccaactgcaa gaaaaccctg gaatgcacca agctgtgcct
>........................................CRD4.........................................>
a g f
f l r
e n e c
v s c
s n c
k k t l
e c t
k l c
gccccagacc gagaacgtga agggcaccga ggacagcggc accacagcgg ccgcagacaa aactcacaca tgcccaccgt
>.>>>>...................stem...................>>
>>.........hinge..........>
l p q t
e n v
k g t
e d s g
t t
d
k t h t
c p p
117
Kirstin A. Zettlitz
Acknowledgements
7 Acknowledgements
First of all, I am very grateful to my supervisor Roland Kontermann: thank you for arousing my
interest in antibody engineering, which really feels like coming into my own. I consider the nearly five
years in your lab to be a crucial learning period. Without your advice and support this thesis would
not have been written.
The first year of my PhD thesis project was done in collaboration with Gabriele Multhoff (München,
Germany) and I have to thank for providing cmHsp70.1 and Colo+ cell line. Thanks to Julia Seitter: A
problem shared is a problem halved. I would like to thank Miriam Rothdiener (Stuttgart, Germany)
for help with the HPLC analysis, and Thomas Johannson (Attana, Stockholm, Sweden) for help in
affinity measurements. This work was funded by a grant from the BMBF (BioChancePlus 0313686B).
Special thanks are due to Roland and Klaus Pfizenmaier to admit me to the ATROSAB project after
the Munich collaboration expired, that was a real life-saver. And I wish to extend special thanks also
to Peter Scheurich: thanks to all three of you for tirelessly discussing my experimental results and my
countless TNF-questions.
I thank Sabine Münkel, who cloned the IZI-06.1 derivatives and I show appreciation to all the people
at Celonic (Jülich, Germany and Basel, Switzerland) who contributed to this project. Thanks to Verena
Lorenz for producing and purifying the different proteins. Special thanks are also due to Andreas
Herrmann for reading each of my posters, presentations and this thesis. This work was supported by
a grant from EC FP6, Project NeuroproMiSe, contract # LSHM-CT-2005-018637.
I wish to extend very special thanks to Dafne Müller, for performing the flow cytometry experiments
for the PMNs and especially for her interest and support in my work. Dafne, I appreciated your help
and suggestion. Furthermore I thank Vanessa Kermer for isolation of PBMCs. Special thanks are also
due to Nadine Fuß for her excellent technical assistance in size exclusion chromatography and her
help with cloning and production of scFv. The long lab days were brightened up by all the former and
present “Kontis”. Sylvia, thanks for your company in our addiction to sweets and for discussing every
episode of various TV series. I also loved drinking Sekt with you and Miriam. Little Roland, the many
internet researches have been so much fun. Ronny, thanks for sharing food (Müsli) with me. I also
thank all the other people for the good working atmosphere at the institute.
Finally, I want to express my deep gratitude to my family for their love, support and encouragement.
Very special thanks go to my mother Hannah and my grandmother Margarete, both are shining
examples of strong and intelligent women and inspire me every day.
118
Kirstin A. Zettlitz
CV
8 Curriculum Vitae

Kirstin A. Zettlitz
date of birth:05 October 1976
address: Sonnenblumenweg 3, D-65201 Wiesbaden, Germany
mobile: +49 179 2914731
e-Mail: [email protected]
[email protected]
Experience
University of Stuttgart, Germany
April 2007 – July 2010
Doctorate at the Institute of Cell Biology and Immunology, Biomedical Engineering,
Allmandring 31, D-70569 Stuttgart, Germany

Doctorate at the group of Roland E. Kontermann. PhD thesis title: “Engineered
Antibodies for the Therapy of Cancer and Inflammatory Diseases”.
Humanization and characterization of a mouse monoclonal antibody specific for
membrane-bound heat shock protein 70 (Hsp70) for cancer therapy.
Epitope mapping, affinity maturation and functional characterization of a humanized
Tumor Necrosis Factor 1 (TNFR1)-specific antibody for therapy of inflammatory diseases.
Education
University of Stuttgart, Germany
April 2007, Diplom in Biology (technical orientation), overall grade: excellent (1.1)
May 2006 – April 2007

Diploma thesis at the group of Roland E. Kontermann, Institute of Cell Biology and
Immunology. Thesis title: “Recombinant Hsp70-specific Antibodies for Cancer Therapy”,
grade: excellent (1.0).
September 2005 – April 2006

Student research project at the group of Roland E. Kontermann, Institute of Cell Biology
and Immunology. Thesis title: “Production and Characterization of modified single-chain
Fv Antibody Fragments”, grade: excellent (1.0).
October 2000 – April 2006




Main study period in technical biology.
Major subject: Antibody Engineering, grade: excellent (1.0).
Minor subjects: Industrial Genetics, grade: good (1.7); Microbiology, grade: excellent
(1.0).
Compulsory optional subject: Biochemistry, grade: excellent (1.3).
119
Kirstin A. Zettlitz
CV
September 1997 – September 2000

Basic study period, Intermediate examination, overall grade: good (2.2).
October 1996 – April 1997
Internship: municipal waterworks laboratory, ESWE, Wiesbaden, Germany
Martin-Niemöller-Schule, Wiesbaden, Germany
1993 - 1996, Abitur (equivalent to High School Exam)
Helene-Lange-Schule, Wiesbaden, Germany
1987 - 1993, comprehensive secondary school
Blücherschule, Wiesbaden, Germany
1983 – 1987, elementary school
Further Education


Certificate Laboratory Animal Science: lecture on Laboratory Animal Science and
practical course on experimental techniques at the Department of Animal Physiology,
Institute of Biology (July 2007).
Wall Street Institute, School of English, Diploma Advanced Stage, certificate equivalent to
BULATS score of 60 (September 2005).
Publications







Zettlitz KA, Seitter J, Muller D and Kontermann RE. Humanization of a Mouse Monoclonal
Antibody Directed Against a Cell Surface-Exposed Epitope of Membrane-Associated Heat
Shock Protein 70 (Hsp70). Mol Biotechnol. 2010.
Zettlitz KA, Lorenz V, Landauer KH, Muenkel S,Herrmann A, Scheurich P, Pfizenmaier K
and Kontermann RE. ATROSAB, a humanized antagonistic anti-tumor necrosis factor onespecific antibody. mAbs 2010.
Stork R, Zettlitz KA, Muller D, Rether M, Hanisch FG, Kontermann RE. N-glycosylation as
novel strategy to improve pharmacokinetic properties of bispecific single-chain
diabodies. J Biol Chem 2008;283:7804-7812.
Hopp J, Hornig N, Zettlitz KA, Schwarz A, Fuss N, Muller D and Kontermann RE. The
effects of affinity and valency of an albumin-binding domain (ABD) on the half-life of a
single-chain diabody-ABD fusion protein. Protein Eng Des Sel 2010.
Muller D, Trunk G, Sichelstiel A, Zettlitz KA, Quintanilla M, Kontermann RE: Murine
endoglin-specific single-chain Fv fragments for the analysis of vascular targeting
strategies in mice. J Immunol Methods 2008;339:90-98.
Zettlitz KA, Generation of heavy and light chains (chimeric antibodies). In: Antibody
Engineering Vol.1, Kontermann, RE and Dübel, S (eds.), Springer-Verlag Berlin Heidelberg
2010.
Zettlitz KA, Protein A/G Chromatography. In: Antibody Engineering Vol.1, Kontermann,
RE and Dübel, S (eds.), Springer-Verlag Berlin Heidelberg 2010.
Communications




PEGS Protein Engineering Summit, Boston 2010, poster presentation.
IBC: Antibody Engineering, San Diego 2009, student poster scholarship.
PEGS Europe: Protein Engineering Summit, Hannover 2009, poster presentation.
CNIO: Recombinant Antibodies: new developments for future challenges, Madrid 2008,
poster presentation.
120

Engineered Antibodies for the Therapy of Cancer and Inflammatory

Transcrição

Documentos relacionados

Company Profile Biotecnol is a biopharmaceutical

Rabbit Polyclonal Antibody to Human Cadherin

Antibody Engineering

Immunisation with foamy virus Bet fusion proteins as novel strategy

Monroe: Coagulation Factor Interaction with Platelets

European Innovative Training Network IMMUTRAIN 15 positions as

Supporting Information - Royal Society of Chemistry

as a PDF

BINDINGS

Dual Antigen-Restricted Complementation of a

Lyme disease in dogs - Borreliose Nachrichten

Update: State of the Art-Borreliose Diagnostik - BCA

Dissection of the IgE Interactome on a Molecular Level