Reconstruction and biologic activity of mouse monoclonal
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UNIVERSITY OF TARTU FACULTY OF SCIENCE AND TECHNOLOGY INSTITUTE OF MOLECULAR AND CELL BIOLOGY DEPARTMENT OF MICROBIOLOGY AND VIROLOGY Aleksei Suslov Reconstruction and biologic activity of mouse monoclonal antibodies inhibiting RNA-dependent RNA-polymerase of the Hepatitis C Virus Master of Science thesis Supervisor: Andrei Nikonov, M. Sc. Department of Biomedical Technology, Institute of Technology, University of Tartu TARTU 2010
Transcript of Reconstruction and biologic activity of mouse monoclonal
UNIVERSITY OF TARTU
FACULTY OF SCIENCE AND TECHNOLOGY
INSTITUTE OF MOLECULAR AND CELL BIOLOGY
DEPARTMENT OF MICROBIOLOGY AND VIROLOGY
Aleksei Suslov
Reconstruction and biologic activity of mouse
monoclonal antibodies inhibiting RNA-dependent
RNA-polymerase of the Hepatitis C Virus
Master of Science thesis
Supervisor: Andrei Nikonov, M. Sc. Department of Biomedical Technology,
Institute of Technology,
University of Tartu
TARTU 2010
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Table of contents
ABBREVIATIONS ............................................................................................................................................... 4
INTRODUCTION ................................................................................................................................................ 6
1. BACKGROUND ......................................................................................................................................... 9
1.1. ANTIBODY STRUCTURE AND FUNCTION ............................................................................................ 9
1.1.1. Antibodies – primary effector molecules in humoral immunity ........................................ 9
1.1.2. Antibody structure .......................................................................................................... 10
1.1.3. Organization of immunoglobulin genes .......................................................................... 13
1.1.4. Fine structure of variable regions. .................................................................................. 16
1.1.5. Canonical structures ........................................................................................................ 17
1.2. DESIGNING ANTIBODIES FOR THERAPY ........................................................................................... 19
1.2.1. Chimeric and humanized mAbs ....................................................................................... 19
1.2.2. Intracellular antibodies (intrabodies) .............................................................................. 21
2. EXPERIMENTAL PART ............................................................................................................................ 24
2.1. AIMS OF THE THESIS .................................................................................................................. 24
2.2. METHODS ............................................................................................................................... 24
2.2.1. Plasmids, bacterial strains and media. ........................................................................... 24
2.2.2. Total RNA isolation with TRIzol reagent ......................................................................... 25
2.2.3. Cloning procedures: ........................................................................................................ 26
2.2.3.1. Reverse Transcription-Polymerase Chain Reaction (RT-PCR) ................................................... 26
2.2.3.2. Restriction Digestion of DNA (RD) ............................................................................................ 27
2.2.3.3. Ligation ..................................................................................................................................... 28
2.2.4. DNA sequencing and analysis. ........................................................................................ 28
2.2.5. Plasmid construction ....................................................................................................... 28
2.2.5.1. Site-directed mutagenesis ........................................................................................................ 28
2.2.5.2. Restriction-free cloning. ........................................................................................................... 30
2.2.5.3. Design and construction of chimeric 7G8 mAb genes .............................................................. 32
2.2.5.4. Design and construction of SapI vector system. ...................................................................... 32
2.2.5.5. Generation of F-2A bicistronic constructs ................................................................................ 33
2.2.5.6. Grafting of 7G8M FR1 regions to 7G8H antibody..................................................................... 34
2.2.6. Production and purification of recombinant mAbs ......................................................... 34
2.2.7. HCV RdRp purification ..................................................................................................... 36
2.2.8. Protein analysis and quantification methods .................................................................. 36
2.2.8.1. Protein quantification using Bradford protein assay ................................................................ 36
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2.2.8.2. Western blot ............................................................................................................................ 37
2.2.9. Enzyme-linked Immunosorbent Assay (ELISA)................................................................. 37
2.2.10. Changing mAb buffer by ultrafiltration .......................................................................... 38
2.2.11. Primer-dependent RdRp assays ...................................................................................... 38
3. RESULTS AND DISCUSSION .................................................................................................................... 40
3.1.1. Cloning of antibody variable regions .............................................................................. 40
3.1.2. Construction of chimeric 7G8 antibody genes ................................................................ 42
3.1.3. Generation of a vector system for high-throughput construction of recombinant
antibody genes. ............................................................................................................... 44
3.1.4. Construction of a panel of recombinant 7G8 and 8B2 monoclonal antibodies. .............. 48
3.1.5. Design and construction of bicistronic mAb expression vectors utilizing the F-2A peptide
sequence. ........................................................................................................................ 49
3.1.6. Expression and purification of enzymatically active NS5B polymerase. ......................... 52
3.1.7. Biologic activity of recombinant 7G8 and 8B2 mAbs ...................................................... 53
3.1.7.1. Biologic activity of recombinant mAbs in antigen binding assay (ELISA).................................. 54
3.1.7.2. Biologic activity of recombinant mAbs in primer dependent RdRp assay. ............................... 55
3.1.8. Identification of mutations, possibly enhancing the biologic activity of 7G8M mAb. ..... 59
3.1.9. Exchanging FR1 regions of 7G8H antibody for those of 7G8M. ...................................... 60
SUMMARY ..................................................................................................................................................... 61
RESÜMEE ....................................................................................................................................................... 63
ACKNOWLEDGEMENTS .................................................................................................................................. 65
REFERENCES ................................................................................................................................................... 66
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Abbreviations
(+)ssRNA single-strand RNA of positive polarity
Ab antibody
ADCC antibody-dependent cellular cytotoxicity
ATP adenosine triphosphate
bp basepair
BPV-1 Bovine Papilloma Virus type-1
BSA bovine serum albumin
CDR complementarity-determining region
CIAP calf intestine alcaline phosphatase
CLL chronic lymphocytic leukemia
CMV Cytomegalovirus
cpm counts per minute
CTCL cutaneous T-cell lymphoma
CTP cytidine triphosphate
DEPC diethylpyrocarbonate
DTT dithiotreitol
EBV Epstein-Barr Virus
ECL enhanced chemiluminescense
EGTA ethylene glycol tetraacetic acid
ELISA enzyme-linked immunosorbent assay
ER endoplasmic reticulum
F(ab) fragment, antigen binding
F(c) fragment, crystallizable
FDA Food and Drug Administration
FMDV Foot-and-Mouse Disease Virus
FR framework
GF/C glass fiber/cellulose
GMP guanosine monophosphate
GTP guanosine triphosphate
HAMA human anti-mouse antibodies
HC heavy chain
HCV Hepatitis C Virus
HPLC high performance liquid chromatography
HRP horseradish peroxydase
IACT Intracellular Antibody Capture Technology
Ig immunoglobulin
IPTG isopropyl-b-D-thiogalactoside
LC light chain
mAb monoclonal antibody
M-MuLV Moloney Murine Leukemia Virus
NFDM non-fat dry milk
Ni-NTA nickel-nitriloacetic acid
NLS nuclear localization signal
NS3 non-structural protein 3
NS5B non-styructural protein 5 B
nt nucleotide
5
NTP nucleotide triphosphate
OD optical density
ORF open reading frame
PBS phosphate buffered saline
PVDF polyvinylidene fluoride
RD restriction digest
RdRp RNA-dependent RNA-polymerase
RSV Respiratory Syncytial Virus
RT reverse transcriptase
RT-PCR reverse transcription polymerase chain reaction
scFv single-chain fragment variable
SDM site-directed mutagenesis
SDS sodium dodecyl sulfate
SDS-PAGE SDS-polyacrilamide gel electrophoresis
TBSV Tomato Bushy Stunt Virus
TCA trichloroacetic acid
UTP uridine triphosphate
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Introduction
Antibodies (Abs), or immunoglobulins, are Y-shaped protein molecules capable of
recognition and discrimination of hundreds of millions of self and nonself substances (termed
antigens) in a host organism. Antibodies are the central component of adaptive immune
system and can be found on the membrane of B-cells (as B-cell receptors (BCR)), or in blood,
in a secreted soluble form [1]. All BCR molecules on the membrane of a single B-cell are
identical, making them specific to only one antigen. After first encounter with its antigen, B-
cell can differentiate into plasma cell and start secretion of large amounts of the same
antibody counteracting the invading microorganism. This might happen only when specific
costimulatory signals are present and antigen is identified as non-self entity by innate immune
system component (for review see Janeway 1989 [2]).
Antibodies are heterodimeric molecules, typically consisting of four polypeptide
chains – two identical heavy chains (~50 kDa) and two identical light chains (~25 kDa) [3-5].
Each light chain is linked to a heavy chain, and the heavy chains are linked to each other by
disulfide bonds [4]. The N-terminal parts of heavy and light chains (the arms of the Y-shaped
molecule) are termed as F(ab) (fragment, antigen binding) and contain regions of high
variability in amino acid sequence [6]. These regions are known as “variable domains” (VH
for heavy chain, and VL for light chain). Each B-cell clone expresses antibody with variable
domains different from those of any other antibody, expressed by any other B-cell clone. Each
variable domain contains unique complementarity determining regions (CDR). The CDRs of
both heavy and light chains form loops of various conformations and interact directly with the
antigen [7-8]. The remaining parts of the heavy and light chains are termed as constant
regions. Constant domains of the antibody heavy chains (constituting the stem of the Y-
shaped molecule) are called F(c) region (fragment, crystallizable). The F(c) region triggers
different effector functions (such as activation of complement system, phagocytosis, cell lysis
by cytotoxic lymphocytes) – cellular mechanisms aimed at elimination of antigens [1, 9].
The great diversity of the antibody repertoire allows isolation of mAbs against
virtually any possible antigen. Due to their exquisite specificity and high affinity to a
particular antigen, antibodies are of great interest for therapeutical applications. After
introduction of hybridoma technology (generation of immortal hybrid cells from myeloma
and mAb-producing B-cell) [10] a lot of murine mAbs with therapeutic potential was
generated and launched into clinical trials. Unfortunately, the vast majority of these mAbs
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failed, because they were ineffective in activation of effector functions in humans. Induction
of the anti-immunoglobulin response, which triggered rapid clearance of mAbs from patient’s
blood was one of the primary issues associated with antibodies deployment as therapeutics.
Such anti-mAb response was termed human anti-mouse antibodies response (HAMA).
Further attempts to make the antibodies less immunogenic and more effective were
undertaken mostly in two directions. First, human hybridomas were generated. Second, mAb
sequence was modified to make it more “human”-like, while maintaining its antigen-binding
specificity. The latter technique included generation of chimeric mAbs in which murine
variable regions were joined to human constant regions (chimerization), and humanization of
antibodies by reshaping its surface (grafting only the CDRs from murine antibody to an
antibody of a human source). Chimeric and humanized mAbs proved to be effective in
clinical studies also reducing HAMA response, at least in some cases. Development of fully
human antibody production technologies (such as phage display and transgenic mice) in
1990-s has lessened the interest towards chimerization and humanization of murine mAbs.
However, humanized antibodies constituted approximately half of all mAbs being in phase 3
clinical studies in 2005 [11].
Further advances in genetic engineering techniques allowed the use of antibodies in a
new format, such as antigen-binding fragments (Fab) or single-chain fragment variable (scFv)
molecules, while retaining the antigen-binding properties of the original mAb. scFv molecules
represent the VH and VL domains of an antibody, joined together with a flexible amino acid
linker. If correctly designed, such molecules do not need the intra-domain disulfide bonds and
can fold properly in the reducing cellular environment and function inside the cell [12].
Antibodies or antibody fragments that perform their functions inside the cell – in cytoplasm,
nucleus or endoplasmic reticulum (ER) – are called intracellular antibodies, or “intrabodies”.
Intrabodies functioning in ER are also termed as “retained antibodies”, because they contain a
ER retention signal. Due to their small size and ability to function inside cell, scFv antibodies
have gained popularity during the last two decades, stimulating the development of
technologies aimed at isolation of functional intrabodies from large recombinant scFv
libraries.
The theoretical part of this study is dedicated to antibody role in immunity, its
structure-function relationship, therapeutic potential of monoclonal antibodies and several
technologies used for design of therapeutical antibodies and intrabodies.
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Experimental part describes cloning of antibody variable domains, construction of
chimeric mouse/human and mouse recombinant antibodies, expressed whether from one or
two promoters, and biologic activity of these recombinant constructs compared to their
hybridoma counterparts.
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1. Background
1.1. Antibody structure and function
1.1.1. Antibodies – primary effector molecules in humoral immunity
Antibodies are glycoproteins, which belong to immunoglobulin superfamily, and are
frequently called immunoglobulins. Antibodies can recognize and bind an antigen – a foreign
substance that can be introduced to an organism for example during viral or microbial
infection. Antibodies bind their antigens with very high affinity, at the same time being highly
specific. Adaptive immune system has and is capable of generating large repertoire of
antibodies specific for various antigens. These immunity properties are vital for the host
organism survival.
Antibodies are expressed by mature B-lymphocytes initially in a form of a membrane
receptor, or B-cell receptor (BCR) [1]. When a B-cell first time encounters an antigen that
matches its BCR, the antigen binding together with proper costimulatory signals (for review
see Janeway 1989 [2]) induces several signal transduction pathways, which result in
activation of B-cell – expression of genes responsible for augmentation of its immunological
functions. After receiving of additional signals from T-helper cells, the B-cell starts to
proliferate (clonal expansion) and its progeny differentiates into plasma cells or into memory
cells [13-14]. Memory B cells have longer life span than the naïve B-cell (one that has not
previously encountered antigen) and express the same membrane-bound antibody as their
parent B-cell. These cells are needed for fast induction of immune response in case of
recurrent introduction of the same antigen. Plasma cells, on the contrary, live only for few
days, but during that time they produce an enormous amount of a secreted form of the
antibody. Secreted antibodies are the major effector molecules of humoral immunity. They
circulate in body fluids, where they find the antigen and neutralize it by blocking its activity,
or, bound to the antigen, activate various effector functions that mediate the clearance of the
antigen from the organism.
Antigens are usually complex molecules (with the exception of haptens) and contain
multiple structures on their surface where antibodies can bind. The part of the antigen
recognized by an antibody is called “epitope” or “antigenic determinant”. Epitopes can be
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linear or conformational. In case of a peptide antigen, linear epitope is a continious stretch of
amino acids, whereas conformational epitope is a particular three-dimensional structure on the
surface of the antigen, that can be formed from different parts of the polypeptide chain.
Normally, one antibody can recognize only one epitope. Consequently, different antibodies
generated during immune response may bind to different epitopes of the antigen, without
interferring with each other.
Each plasma cell expresses a unique antibody during its whole life cycle. Such
antibody is termed as monoclonal antibody. Polyclonal antibodies are mixture of monoclonal
antibodies secreted by single type B-cells in response to a specific antigen. Polyclonal
antibodies recognize different epitopes on the same antigen.
1.1.2. Antibody structure
The name “immunoglobulins” is derived from the initial finding that antibodies
migrate with globular proteins during the electrophoresis of the antibody-containing serum
[15]. By structure, antibodies are heterodimeric multichain glycoproteins typically consisting
of four peptide chains: two identical heavy chains (HC) of ~50 kDa molecular weight, and
two identical light chains (LC) of ~25 kDa molecular weight (Figure 1) [3-5]. One light and
one heavy chain form a heterodimer, held together by covalent disulfide (S-S) bond [4], and
several noncovalent interactions. These light-heavy chain complexes in turn dimerize to form
a functional Y-shaped antibody molecule (Figure 1). Heavy chains of these LC-HC
heterodimers are also linked to each other by disulfide bridges and noncovalent interactions
[4]. The „arms“ of the molecule are responsible for the antigen binding and are called F(ab)
fragments (Figure 1) (fragment, antigen binding) [16-17]. The “stem” of this Y-like structure
is called F(c) fragment (Figure 1) (fragment, crystallizable). It is capable of binding to
specific receptors on a cell membrane and is responsible for antibody effector functions, for
example, activation of proteins of the complement system [9]. These structural and functional
properties were established by proteolytic cleavage of immunoglobulins with pepsin or papain
(Figure 2) and examination of the biological activity of obtained products [9, 16-20].
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Figure 1. Schematic diagram of immunoglobulin structure. N-terminal portions of both chains are variable
regions (VH and VL), which are responsible for antigen binding. The remainders of the molecule are constant
regions, which define the antibody isotype and subclass (for light chain). Hinge region providing flexibility to
antigen-binding regions is drawn in black. The region below hinge is called Fc and it is responsible for activation
of effector functions. Figure and legend source: Kuby Immunology 5-th edition [1].
The N-terminal fragments (~110 aa in length) of both heavy and light chains, the
“tips” (or the “hands”) of the Y-shaped molecule arms, exhibit great sequence diversity
between antibodies of different specificities. These regions are called variable regions (or V
regions) and designated as VH and VL for heavy and light chain, respectively (Figure 1) [1].
The remaining parts of the antibody chains are termed as constant regions (CH and CL).
Constant regions of heavy chains consist of three (or four) structurally conserved constant
domains – CH1, CH2, CH3 (and CH4) [1]. Light chains contain only one constant domain,
adjacent to the variable region. The CH1 domain of the constant chain (juxtaposed to the VH
region) associates with CL domain of the light chain, and together they serve as a stable basis
for paired VH and VL domains, which contain antigen-binding sites.
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Figure 2. Prototype structure of IgG, showing disulfide bonds. Fragments produced by various treatments
(pepsin, papain) are also indicated. Figure and legend source: Kuby Immunology 5-th edition [1].
Based on the amino acid sequences of their constant regions, antibody heavy chains
are classified into five groups, called isotypes – α, µ, γ, δ, and ε. Within the bounds of a
particular isotype, sequences of constant chains are very conserved, with the exception of α
and γ isotypes, which can have minor differences and therefore are divided into subisotypes
(γ1, γ2, etc.). The type of the heavy chain defines the class of the immunoglobulin molecule –
IgA(α), IgG(γ), IgM(µ), IgD(δ) or IgE(ε). IgG and IgA class antibodies are divided into
subclasses, depending of the subisotype of the heavy chain. In humans, there are 2 different
subisotypes for α chain, and 4 subisotypes for γ chain. The respective immunoglobulin
subclasses are IgA1, IgA2, IgG1, IgG2, IgG3 and IgG4. For light chains there are only two
types of constant regions – kappa (κ) and lambda (λ). On the basis of the few amino acid
substitutions, λ chains are divided into subtypes - λ1, λ2, λ3, (λ4) - 4 subtypes in humans and
3 subtypes in mice. Each immunoglobulin class can have either κ or λ light chain, but never
both in the same molecule. Heavy chains that consist of three CH domains also have a
proline-rich spacer region – hinge - between CH1 and CH2 domains (Figure 1). Hinge is very
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flexible and allows a wide-range motion of a F(ab) fragment relative to both F(c) and to the
other F(ab) [21-22].
Immunoglobulin variable regions are homologous in overall structure among different
antibodies, but are very diverse in their amino acid sequence in antibodies of different
specificities, with the major part of this diversity falling into the sequences of the antigen-
binding sites. At the same time constant regions are very conserved in their sequence and can
be divided into small number of different types and subtypes. Due to this diversity in variable
regions, very large repertoire of antibodies can be generated in a single organism. The reason
for this is very simple: antigens can be very different in structure, and in order to be effective
against all of them, the immune system should have huge enough number of different
potential antigen binders prior to antigen exposure, so that virtually any antigen could be
bound by pre-existing antibodies as soon as possible. At the same time each antibody has to
be capable of activating series of effector functions. This explaines the limited number of
constant region sequences and their conservation. Summarizing all of the described features
of antibody structure, it becomes clear that these molecules are designed in such a way, that
they could be on one hand easily adjusted to rapidly changing conditions (introduction of new
pathogens, or mutation of old ones) and on the other hand could always perform the basic
activatory functions. Mechanism for generation of this very interesting duality should be
different from mechanism for generation the majority of other molecules, but at the same time
evolutionarily conserved on a gene level. The organization of immunoglobulin genes
completely explains this phenomenon.
1.1.3. Organization of immunoglobulin genes
Each type of the antibody chains (the κ and λ light chains and the heavy chains) is
encoded by different gene families, which locate in separate chromosomes. These gene
families are also known as multigene families, because they consist of multiple gene segments
of different types (Figure 3). The gene segments are rearranged with each other in the process
of B-cell maturation by means of recombination. Only one segment (from the pool of multiple
sequences) of each type is present in a final gene that is transcribed into mRNA and expressed
as the immunoglobulin chain. The phenomenon of this DNA rearrangement was first
discovered by Tonegawa in 1976 [23]. His group isolated genomic DNA from mouse embryo
cells and from differentiated plasmocytoma cells (type of B-cell cancer, that produces κ-light
chains), digested the DNA with restriction enzyme and analyzed the products by Southern
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blot method, using a radioactively labelled κ-light chain mRNA and its 3’-prime end half as
probes. Hybridization patterns for the genomes of embryo cells and plasmocytoma cells were
completely different. In case of rearranged DNA (plasmocytoma) one large fragment
hybridized to both probes, whereas in germ-line (embryo cells) two smaller fragments were
identified, from which one hybridized to both probes, and another to only full mRNA probe.
From the results of this experiment it was deduced, that sequences coding for variable and
constant regions are located separately in genomic DNA, and joined together to create the
continious gene in differentiating lymphocytes [23]. This hypothesis was confirmed in
consequent studies [24-26]. The sequencing of corresponding chromosomal regions further
proved the “multigene” structure of the immunoglobulin genes and allowed to define more
precisely their organization, quantity and spatial distribution [27-28].
Figure 3. Organization of the immunoglobulin germ-line gene segments in the mouse: (a) λ-light chain, (b) κ-
light chain and (c) heavy chain. The light chains are encoded by V, J and C gene segments. The heavy chain is
encoded by V, J, D and C gene segments. The distances in kilobases (kb) between different segments are shown
below each chain diagram. Figure and legend source: Kuby Immunology 5-th edition [1]
Now it is known, that the heavy chain gene family contains four different types of
segments – V (variable), D (diversity), J (joining) and C (constant) and the light chain family
– only three types: V, J and C (either κ or λ) (Figure 3). During the somatic recombination,
these fragments are joined together into one continious transcriptional unit (Figure 4).
Rearranged VDJ fragment corresponds to the heavy chain variable region, and VJ fragment –
to the variable region of the light chain. The C fragments encode the respective constant
regions. All of the known Ig gene families also contains a short sequence upstream to each V
region gene, coding for 19 aa long leader (or signal) peptide (L), which is required for the
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transport of the newly synthesized peptide to the endoplasmatic reticulum (ER) where it
undergoes the correct assembly and glycosylation. Before the antibody is secreted or sent to
the membrane of the B-cell, the signal peptides are cleaved from both H and L chains, and
assembled antibody molecule does not contain any leader sequences.
Figure 4. The process of the heavy chain gene rearrangement and RNA processing. A DH to JH and VH to DHJH
joinings are necessary to generate functional heavy chain gene. Rearrangement of light chains is analogous,
except the absence of D segment. Each C gene is drawn as a single coding sequence; in reality, each is organized
as a series of exons and introns. Rearrangement of the light chain gene occurs similarly. Figure and legend
source: Kuby Immunology 5-th edition [1]
During the process of somatic recombination, first, assembly of V-(D)-J fragments
takes place, then, following transcription and mRNA splicing, VDJ or VJ fragment is joined
to the corresponding C segment. The sequences coding for the constant regions are presented
by only one gene segment for each isotype of heavy chain, one segment for Cκ chain and 4
segments for the λ constant chain. This fact explains the constancy of these regions in all
antibodies. The number of V, (D) and J segments is much higher. For example, human heavy
chain variable regions contain 44 different VH segments with an open reading frame [27], 27
D segments [29] and 9 J segments [30]. By recombination of these segments a huge number
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of rearranged genes can be generated. Further diversification of the antibody repertoire
involves a process known as somatic hypermutation, during which mutations are introduced at
a high rate into the VH and VL coding regions of rearranged immunoglobulin genes [31].
Taken together, both the somatic recombination and hypermutation contribute greatly to the
antibody diversity of a single organism.
1.1.4. Fine structure of variable regions.
Although, the sequences of variable regions can be very different, the overall structure
of these domains is conserved. Analysis of antibody molecule structure by x-ray
crystallography [7, 32-39] revealed that immunoglobulin domains are packed into a particular
type of protein structure known as the immunoglobulin fold. Each domain contains two β-
pleated sheets, which pack face to face ~10 Å apart from each other, with the angle of 30°
between them (Figure 5a). These β-pleated sheets are linked by a conserved disulfide bridge
and consist of antiparallel β-strands (Figure 5a), which are stabilized laterally by hydrogen
bonds and are terminally connected by loops of various length (Figure 5a, 5b). In variable
domains these loops are encoded by the 6 hypervariable regions (CDRs) – 3 in VH and 3 in
VL – and form the antigen-binding site (Figure 5a, 5b). The loops are designated as H1, H2
and H3 for VH domain, and L1, L2 and L3 for VL domain.
Figure 5. A) The schematical structure of immunoglobulin VL domain. Strands of β-sheet are epresented by
ribbons. Hypervariable loops are labelled as L1, L2 and L3. L2 and L3 are hairpin loops that link adjacent β-
sheet strands. L1 links two strands that are part of different β-sheets. The VH domain and its hypervariable loops
H1, H2 and H3 have homologous sequences. B) The arrangement of the 6 hypervariable regions that form the
antibody binding site. The squares indicate the position of residues at the ends of the β-sheet strands in the
framework regions. Figure and legend source: Chothia and Lesk (1987) [8].
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First attempts to correlate the amino acid sequences of variable domains with their
possible structure and function were made by Kabat and colleagues in 1970 [6]. As more and
more sequence data for variable regions were appearing, they could be compared in order to
reveal the structure-function relationship of these domains, responsible for antigen binding.
To systematize the sequence data available for V regions at that time, Kabat’s group created a
database of the aligned amino acid sequences of light chain variable regions and analyzed the
amino acid variabilities at each position. As a result, they have identified three regions of
maximal variation [6]. When the database was supplemented with sequences of heavy chain
variable regions, the same pattern of variability in these sequences was observed. These
hypervariable segments were originally called hypervariable regions and were suggested to
be the antigen-binding sites of the antibody molecule, what was later proved by x-ray
crystallographic studies [7, 32-39]. They correspond to the loops that connect the β-strands
and make direct contact with the antigen. These loops are designated as H1, H2, H3 and L1,
L2, L3 for VH and VL domains, respectively. Because the antigen-binding site is
complementary to the antigen structure, these hypervariable regions are also called the
complementarity-determining regions (CDR). The remainders of the variable region
sequences are known as framework regions (FR) and make up a backbone to which the
hypervariable loops are attached. The framework regions have much lesser variation in amino
acid sequences. They provide the rigid scaffold necessary for maintaining the structural
integrity of the molecule. Several amino acid residues at specific positions were identified,
which can also play important role in CDR conformations [40-43]. This means that despite
main variability in antigen-binding sites is generated by hypervariable regions, framework
regions can “fine-tune” the CDR orientation and, consequently, antibody affinity to antigen.
1.1.5. Canonical structures
Another important finding made by Kabat when comparing the sequences of the
hypervariable regions then known, was the discovery of several conserved residues in these
sequences – 13 in VL regions and 7 in VH regions [44]. It was suggested that these residues
might be responsible for the structure of the hypervariable regions, not for specificity. It was
also supposed that these amino acids have certain positions in the antibody variable regions
and are needed to give a specific conformation to the main-chains of the hypervariable loops.
Padlan and Davies analyzed immunoglobulins of known structure and showed, that in several
cases the hypervariable regions of the same size, but with different sequences have the same
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main-chain conformations [45]. Another analysis of immunoglobulin sequences made by
Padlan revealed that some residues in hypervariable regions, and residues buried within the
domains in the known structures, are conserved [46]. In 1987, Chothia and Lesk examined
immunoglobulins with known atomic structures and identified several residues that through
various interaction with each other or ability to assume unusual values of torsion angles, were
primarily responsible for the main chain conformations of the hypervariable regions in those
structures [8]. The conformations are determined by the interaction of a few residues at
certain sites in the hypervariable regions, and, in some cases, in the framework regions.
Subsequent analysis of the sequences of immunoglobulins of unknown structures showed,
that many of them contained at least one set of residues responsible for a specific
hypervariable conformation (from those observed). These observations suggested, that despite
the sequence variability, most of the hypervariable loops in immunoglobulins have a limited
number of main-chain conformations which were called “canonical structures”. Possible
canonical structures were described for all hypervariable regions, except the H3 loop [8, 47].
The sequence coding for this loop is located at the junction of rearranged V, D and J
segments, therefore it has much more structural variety than all other hypervariable regions.
Using this canonical structure model, Chothia and colleagues tried to predict the structures of
the hypervariable regions of the D1.3 antibody prior to the experimental determination. It was
found that this antibody had the hypervariable regions of the same size, as in previously
analized structures, and the same, or similar, amino acids at key positions determining the
type of the canonical structure [48]. The comparison of the predicted structure with the
obtained crystal structure showed that the conformations of 4 of 6 hypervariable regions had
been predicted correctly, demonstrating the efficiency of this model [48].
The fact, that at least five of six hypervariable regions of any antibody have only a
small discrete set of conformations that can be easily predicted is of the utmost importance for
the antibody engineering. Understanding, which key residues affect the main chain
conformations of the hypervariable region, it is possible to transfer the antigen-binding
specificity from one antibody to another, and at the same time reshape the surface of the host
antibody to match it with the donor antibody if it is needed for corect orientation of the
hypervariable loops. This feature can be extremely useful in the engineering of therapeutic
antibodies (will be discussed below).
19
1.2. Designing antibodies for therapy
1.2.1. Chimeric and humanized mAbs
Soon after the discovery of the antibodies, their therapeutic potential became obvious.
Antibodies could help in solving many medical problems, such as organ grafting, cancer
therapy, or treatment of viral and microbial infections. The first report describing the method
allowing to produce monoclonal antibodies in large quantities was received from Köhler and
Milstein in 1975 [10]. The method involved generation of murine hybridomas – cell lines,
produced by fusion of a mAb-producing B-cell with a myeloma cell. Resulting hybrid cells
were immortal and produced high enough amounts of the monoclonal antibodies. Despite the
initial procedure of creating hybridomas was unreliable, the technique made revolution in the
field of immunology and became a general method for production of monoclonal antibodies.
Many mAbs against various antigens were generated since that time aimed to be used as
therapeutics. However, when these murine monoclonal antibodies entered the clinical study,
their limited potential to be used as therapeutics became evident. Murine antibodies were
identified as antigens by patients’ immune system and were rapidly cleared from blood by the
immune response involving the human anti-mouse antibodies (HAMA). They were also
ineffective in activation of effector functions in humans. However, this obstacle did not
diminish interest towards these very promising molecules, but rather stimulated the
development of various methods and technologies for production of therapeutic mAbs. These
methods were aimed whether at a development fully human antibodies using the hybridoma
technology (what was not really successful at the beginning), or at lessening the antibody
immunogenicity through decreasing the percentage of murine sequences in antibody by
changing them to sequences of human origin. Two main techniques in this approach were
chimerization and humanization of murine mAbs.
20
Figure 6. Schematic drawing of murine, chimeric and humanized antibodies. Murine sequences are shown in
blue and human sequences in red. Source: Clark, M (2000) [49].
Chimeric mAbs are antibody molecules that contain variable regions derived from a
murine source, and constant regions derived from a human source (Figure 6). The first reports
concerning the creation of chimeric mAbs and their biologic activity were received from
Morrison et al. [50] and Boulianne et al. [51] in 1984. They have shown that chimeric
antibodies maintain their biologic activity at a level comparable with that of original murine
mAbs. In the animal models it was shown that replacing the murine Fc region could reduce
the host immune response, however chimeric mAbs could still induce the anti-idiotypic
responses (responses against antibody variable regions), although these were delayed and
weaker [52-53]. Despite the chimeric mAbs were superior to murine mAbs in terms of
therapeutic application, the number of chimeras entering the clinical studies starting from
1987, was lower than that for murine antibodies. However, their approval success rate was
much higher than that for murine mAbs. According to the data available on July 2005, there
were 5 chimeric mAbs being used worldwide in medicine for treatment of various diseases
[11].
Chimeric mAbs did not receive a wide spread occurrence probably, because the
strategy of humanization, which was first applied only few years after the first chimeric mAb
construction seemed to be superior to the chimerization. Humanization approach was based
on assumption, that greater percentage of fully human sequences in mAb would provide
greater reduction in HAMA response. In a process of humanization, only those regions that
make direct contact with the antigen (CDRs) are grafted into a framework of a human
antibody (Figure 6). The first report of successfully humanized antibody, which maintained its
functions after the procedure was received in 1986 [54]. The first successfully humanized
21
therapeutic antibody was produced in 1988 by a group of scientists from Cambridge
University [55]. They have humanized the rat antibody CAMPATH-1 (raised against human
lymphocyte proteins) that might have a therapeutic application in treatment of chronic
lymphocytic leukemia (CLL). The humanized mAb had entered the clinical studies and turned
out to be effective in patients with non-Hodgkin’s lymphoma, and at the same time it did not
induce the anti-immunoglobulin response [56]. The humanized CAMPATH mAb was finally
approved as therapeutic by U. S. Food and Drug Administration (FDA) in 2001, and now it is
used under the name Alemtuzumab for the treatment of chronic lymphocytic leukemia (CLL),
cutaneous T-cell lymphoma (CTCL) and T-cell lymphoma [57-58]. This first successful
humanization was followed by many similar works on humanization of various mAbs against
cancerous or viral antigens [59-66]. However, the humanization turned out to be a difficult
process, and many humanized mAbs reported had a reduced affinity to the antigen, compared
to the original mAb [55, 60, 62, 65, 67-68]. As it is been mentioned in “Canonical structures”
section, specific framework residues are responsible for the main chain conformations of
variable loops and it has to be considered during humanization [68-69]. All critical FR
residues should be also transferred from donor mAb to the final constructs. The prediction of
the conformation and identification of all important FR residues requires a thorough analysis
of sequence and structure of donor and host mAbs, as well as a careful choice of the donor
framework. Although, after the humanization the percentage of donor amino acids in acceptor
mAb may be the same as for chimeric mAbs, humanized antibodies are yet less immunogenic,
probably because of the more “human-like” overall structure.
Development of fully human antibody production technologies (such as phage display
and transgenic mice) in early 1990s has lessened the interest towards chimerization and
humanization of murine mAbs. However, humanized antibodies constituted the majority of
mAbs entered clinical studies during the period from 1992 to 2002 years [11]. As of data
available in July 2005, humanized antibodies made up approximately half of all mAbs being
in phase 3 clinical studies [11].
1.2.2. Intracellular antibodies (intrabodies)
Naturally occurring antibodies are extracellular molecules. They are synthesized in
endoplasmic reticulum (ER) and secreted into the blood, where they bind antigens and
activate different effector function via the interaction of the F(c) domain with proteins of the
complement system [9], or with receptors present on the membrane of white blood cells. Even
22
without activation of the effector functions, antibodies are able to neutralize antigens in
different ways by simply binding to them. Abs can prevent various molecular interactions, for
example, block the activity of an enzyme by locking it in an nonfunctional conformation or
blocking its active site(s), or bind to substrate molecules, for instance, viral DNA or RNA,
preventing their access to the enzyme. So, it is reasonable to suppose, that all of these effects,
that could be achieved outside the cell should be achievable inside the cell, as well. This
assumption stimulated the development of intracellular antibodies (or intrabodies). The term
“intrabody” refers to an antibody (or a part of antibody) that functions within the bounds of
the outer membrane of a cell – in the cytoplasm, in the nucleus or in the ER. The first report
confirming that antibody can efficiently function inside a cell was received in 1980, when it
had been shown that after microinjection of purified antibodies into individual cells they
could block the function of the target molecule [70]. Later, it was demonstrated that
functional antibodies could be expressed in mammalian cells, and could be targeted to the
nucleus by utilizing the nuclear localisation signal (NLS) [71]. However, this was rather an
exception. The majority of attempts to express antibodies in cytoplasm had failed, because the
reducing environment of cell cytoplasm prevents the formation of inter- and intradomain
disulfide bridges and, consequently, correct folding of the antibody [72-73]. As activation of
the effector functions is not required from the antibody, functioning inside a cell, “scFv”
(single-chain fragment variable) became the most preferable format for design of intracellular
Abs. scFv is a molecule of ~25 kDa molecular weight, and it is composed of antibody heavy
and light chain variable domains, connected with a flexible amino acid linker (Figure 7). The
adoption of this format removed at least one problem – formation of inter-chain disulfide
bridges, because the domains of the scFv molecule are linked physically, what facilitates their
correct association. However, this technological advance by itself was not enough to generate
stable intrabodies with high expression levels.
Figure 7. Schematic drawing of a typical scFv molecule. VH (blue) and VL (orange) domains are shown,
connected with a peptide linker. N-terminus and C-terminus are designatet as N and C, respectively.
23
Development of a strategy for prediction of stabilizing mutations in immunoglobulin
domains, and implication of a consensus engineering approach (engineering of antibody
domains based on consensus sequences), finally led to the construction of stable scFv
molecules that could be efficiently expressed [74-76]. The approach was based on the analysis
of available immunoglobulin sequence data, determination of consensus sequences and
introduction of stabilizing mutations identified by this analysis. Simultaneously, it was shown,
that scFv possessing a stable framework can fold correctly and function without the disulfide
bond [12]. Further development of in vivo selection strategy for functional intrabodies –
Intracellular Antibody Capture Technology (IACT) – which utilized the modified yeast two-
hybrid screening system [77], allowed isolation of various stable and functional intrabodies
from artificially created recombinant scFv libraries [78-79]. Analysis of their sequences
revealed great similarity of their framework structures and allowed to define the stable
consensus framework that could be used for intrabody engineering and generation of new de
novo intrabody libraries [78-80]. In subsequent experiments it was demonstrated, that grafting
of foreign CDRs into this framework yielded highly stable and functional intrabodies [81].
Thus, intrabody consensus framework seems to be a powerful platform for engineering
intracellular antibodies with various specificities or generation of synthetic intrabody libraries
based on this highly stable sequence.
Starting from 1990, many intrabodies with proved efficiency in blocking
intermolecular interactions was developed (for references see [82-83]). Of particular interest
are several antiviral intrabodies, for example scFv against the NS3 protein of Hepatitis C
Virus (HCV), which is capable of inhibiting the viral replication [84-85], or intrabodies
against RNA-dependent RNA polymerase (RdRp) of Tomato Bushy Stunt Virus (TBSV), that
are capable of protecting plants against the viral infection [86]. The latter intrabodies are also
capable of inhibiting the RdRp-s of heterologous closely related viruses, and even of binding
the HCV RdRp [86]. TBSV and HCV belong to the same supergroup and the replication
strategies of these viruses are similar, what means, that the same approach may work in case
of HCV or other (+)ssRNA virus.
First and last, intrabodies are very promising biomolecules to be used as intracellular
therapeutics. Owing to the development of in vivo intrabody capture technologies the
generation of highly engineered intrabodies is recently becoming a reliable and robust
procedure with great potential and may result in a new wave of therapeutics on the market in
nearest future.
24
2. Experimental part
2.1. Aims of the thesis
The main objective of this study was the construction of various recombinant forms of
mouse monoclonal antibodies 7G8 and 8B2, previously identified in our lab and shown to be
capable of inhibiting the activity of HCV RNA-dependent RNA-polymerase in vitro [87], and
demonstration of their biologic activity. This work is an intermediate step in preparation of
these antibodies to a possible therapeutical use in future. Following specific aims were bound.
First, amplification of variable regions of mAbs 8B2 and 7G8 from hybridoma cDNA.
Second, construction of recombinant mouse/human and mouse/mouse 7G8 and 8B2 genes
using expression vectors (developed by Icosagen Group) with pre-cloned mouse and human
antibody constant regions. Third, development of a high-throughput system for construction
of recombinant antibody genes. Fourth, generation of single-promoter expression constructs
for recombinant 7G8 and 8B2 antibodies, based on Foot-and-Mouse Disease virus 2A peptide
and furin cleavage site. Fifth and sixth, purification of enzymatically active HCV RdRp
(NS5B) for biochemical assays and comparison of the biologic activity of generated
recombinant mAbs to that of original hybridoma 7G8 and 8B2 mAbs.
2.2. Methods
2.2.1. Plasmids, bacterial strains and media.
pJet 1.2/blunt (2974 bp) (Fermentas) - positive selection cloning vector containing a
lethal restriction enzyme gene that is disrupted by ligation of a DNA insert into the cloning
site. Contains the pMB1 rep element responsible for the replication of plasmid, amp
resistance gene, multiple cloning site (MCS). In this work used as an intermediate host for
cloning of antibody variabe regions from hybridoma.
pBluescript II SK+ (Stratagene) - Contains f1 helper phage origin of replication, amp
resistance gene, and MCS within LacZ gene fragment, which allows white/blue screening. In
this work used as an intermediate vector for almost all cloning procedures.
pCMV (Icosagen Group) – Expression vector, that consists essentially of 3 parts –
antibiotic resistance marker (Km), Bovine Papilloma Virus type-1 (BPV-1) E2 or Epstein
25
Barr Virus (EBV) EBNA sequence, which allows this plasmid not to get lost during cell
division, and gene expression cassette(s) (in this case – Ab expression cassettes). Expression
is driven by Cytomegalovirus (CMV) early promoter
pCMV_SV40pA_h-mAb2_42 (here – pCMV42) – pCMV vector with cloned codon-
optimized human IgG1 heavy and kappa light chains
pCMV_SV40pA_m-mAb1_65 (here – pCMV65) - pCMV vector with cloned codon-
optimized mouse IgG1 heavy and kappa light chains
pCMV_SV40pA_m-mAb2_83 (here – pCMV83) - pCMV vector with cloned codon-
optimized mouse IgG2a heavy and kappa light chains
pET-19bNS5b[Con1] (Merck) – Expression vector, containing a HCV NS5B coding
gene, cloned under the control of the bacteriophage T7 promotor. Also contains a
decahistidine tag coding sequence, fused directly to the 5’ end of the NS5B gene.
Bacterial strains:
E.coli DH5α - F- endA1 glnV44 thi-1 recA1 relA1 gyrA96 deoR nupG Φ80dlacZΔM15
Δ(lacZYA-argF)U169, hsdR17(rK- mK
+), λ–. Growth and purification of plasmid DNA.
E.coli BL-21 (DE-3) (Stratagene) - F– ompT gal dcm lon hsdSB(rB
- mB
-) λ(DE3 [lacI
lacUV5-T7 gene 1 ind1 sam7 nin5]). Expression and purification of HCV NS5B polymerase.
All bacterial cultures were grown in Luria-Bertani (LB, Difco) medium (10 g/l bacto-
tryptone, 5 g/l bacto-yeast extract, 10 g/l NaCl) supplied with an appropriate antibiotic at a
final concentration of 100 µg/ml for Amp, and 50 µg/ml for Km.
2.2.2. Total RNA isolation with TRIzol reagent
Total RNA was isolated from hybridoma cell lines using commercially available
ready-to-use TRIzol reagent (Invitrogen). The reagent is a mono-phasic solution of phenol
and guanidine isothiocyanate. TRIzol can disrupt cells and dissolve cell components, at the
same time maintaining the integrity of the RNA. After addition of chlorophorm and
centrifugation, the sample solution is divided into three phases. The upper (aqueous) phase
contains the desired RNA which can be easily recovered by precipitation with isopropyl
alcohol.
Hybridoma cells (~107), producing mAbs 7G8, 8B2 and 10D6 were centrifuged at
270×g for 5 minutes and pellets were homogenized in 1 ml of TRIzol reagent (Invitrogen).
The suspensions were centrifuged for 10 minutes at 12 000×g at +4 °C to remove insoluble
material. After centrifugation supernatants were transferred to new tubes and incubated 5
26
minutes at room temperature to permit the complete dissociation of nucleoporin complexes.
Following incubation, 200 µl of chloroform (per 1 ml of TRIzol) was added to each sample
and tubes were shaken vigorously by hand for 30 seconds. After a 15-min centrifugation at
12000×g at +4°C, the RNA-containing aqueous phase of each sample was transferred to a
new 1.5 ml tube. Further, 500 µl of isopropanol (per 1 ml of TRIzol used for initial
homogenization) was added to each sample and tubes were incubated for 15 min at room
temperature and then centrifuged for 40 min at 16000×g at room temperature (5415R,
Eppendorf) to precipitate and pellet the RNA. Following precipitation, supernatants were
discarded and pellets were washed four times with 1 ml of 70% ethanol (15 min at 16000 ×g
at room temperature). After the last washing step ethanol was discarded and the tubes were
stored opened for 15-20 min at room temperature to remove residual ethanol. Following the
incubation, the RNA was dissolved in 100 µl of RNase-free H2O and additionally incubated
for 30 min at 37 °C in closed tubes to allow complete dissolving of the RNA. RNA yields
were quantified using ND-1000 spectrophotometer (NanoDrop Technologies, Inc.). The
purified RNA yield was up to 180 µg per ~107 hybridoma cells. The 260/230 nm ratio was
2.0, indicationg high enough degree of purity. The integrity of the RNA was analyzed using
denaturing formaldehyde gel-electrophoresis (Figure 11)
2.2.3. Cloning procedures:
2.2.3.1. Reverse Transcription-Polymerase Chain Reaction (RT-PCR)
Reverse transcription of hybridoma total RNA was performed using RevertAid™ First
Strand cDNA Synthesis Kit (Fermentas), that utilises RevertAid™ Reverse Transcriptase
(genetically modified M-MuLV RT). Reactions were performed in a reaction buffer provided
by manufacturer and contained ~1 µg of hybridoma total RNA, 20 units of RiboLock™
RNase Inhibitor, 1 mM dNTP mix, 0.5 µM oligo(dT)18 primer and 20 units of RT.
Diethylpyrocarbonate (DEPC)-treated H2O was added to a total volume of 20 µl.
First, template RNA was mixed with primer in a total volume of 12 µl and denatured
for 15 min at 65 °C. After that, tubes were briefly chilled on ice and all other components
were added. Reaction mixes were incubated for 1 h at 50 °C. Following incubation the
reactions were terminated by heating at 70 °C for 5 min. Reaction products were used directly
for amplification of antibody variable regions by PCR.
27
PCR reactions were performed in a total volume of 50 µl. Reaction mix contained
template DNA (or cDNA), 0.2 µM dNTP mix, 0.5 µM primers, 1x reaction buffer and 1 U of
DNA polymerase – Taq (Fermentas) or Phusion (Finnzymes). During the reaction setup tubes
were constantly held on ice. For Phusion DNA polymerase initial denaturation lasted 30 sec
followed by amplification cycle of 25-35 rounds: 10 sec denaturation at 98 °C, 15-30 sec
primer annealing at 55-72 °C (depending on the annealing temperature of primer) and
extension (time depended on a fragment length, considering sysnthesis rate ~15-30 sec/1000
nt) at 72 °C. If Taq DNA polymerase was used, initial denaturation was performed at 94 for 3
min and during amplification cycles denaturation lasted 1 min at 94 °C. Elongation time was
also increased accordingly to the systhesis rate of Taq polymerase (~1 min/1000 nt).
Reactions were accomplished by final 10 min extension step and stored at -20 °C until
needed.
2.2.3.2. Restriction Digestion of DNA (RD)
DNA digestion reactions were performed using enzymes from Fermentas, according to
manufacturer’s instructions. Enzyme quantity was chosen depending on the amount of DNA.
Generally, 0.5-2 µg of DNA was digested with a 5- to 10-fold excess of enzyme (considering
that 1 unit of enzyme cleaves 1 µg of DNA in 1 h at 37 °C) in a total volume of 20 µl in
appropriate buffer for at least 1 h. When needed, DNA was dephosphorylated after digestion
using Calf Intestine Alcaline Phosphatase (CIAP), (Roche). 3 µl of CIAP (1 u/µl) were added
directly to RD mix and incubated at 37°C for 30 min. The enzyme was inactivated by heating
at 80 °C for 20 min.
Products of PCRs or DNA digestion were resolved on 0.8% agarose gel (or 2%
agarose gel for fragments <200 nt in length), and fragments of correct size were extracted
with „JETquick Gel Extraction Spin Kit“ (Genomed) or „NucleoSpin Extract II Kit“
(Macherey-Nagel) following manufacturer’s recommendations. Alternatively, PCR products
were purified with „JETquick PCR Product Purification Kit“ (Genomed). Purified DNA was
used for downstream applications, such as ligation or PCR.
28
2.2.3.3. Ligation
Ligations occurred in total volume of 20 µl using 5 U of phage T4 DNA ligase
(Fermentas). Approximately 10-20 ng of vector DNA was used per reaction. Molar ratio of
insert to vector was 3 to 1. Reactions were performed at room temperature for 1 h. Up to ¼ of
ligation mix was further used for transformation of chemically competent E.coli DH5α cells.
Bacterial transformation was performed using heat shock method. Transformed cells
were inoculated on LB plates containing an appropriate antibiotic – ampicillin (Amp, final
concentration – 100 µg/µl) (Roche), or kanamycin (Km, final concentration – 50 µg/µl),
depending on a plasmid resistance gene. Plasmid DNA was purified from transformed cells
using NucleoSpin Plasmid QuickPure Kit (Macherey-Nagel), which utilises basic lysis
method. Purified DNA was dissolved whether in Milli-Q water (Millipore) or in Tris-HCl
buffer (pH 8.5).
All cloning procedures were performed exactly as they are described under this
chapter, unless otherwise indicated.
2.2.4. DNA sequencing and analysis.
All constructs generated by PCR were sequenced using sequencing facilities (ABI
3130xl Genetic Analyzer and ABI 3730xl DNA Analyzer) provided by Institute of Molecular
and Cell Biology. Multiple sequence alignments were performed using ClustalW [88]
program provided on European Bioinformatic Institute (EBI) server
(http://www.ebi.ac.uk/Tools/clustalw2/) and analyzed with BioEdit 7.0 software (Tom Hall,
Ibis Therapeutics).
2.2.5. Plasmid construction
2.2.5.1. Site-directed mutagenesis
For site-directed mutagenesis (SDM) of pBSK internal SapI restriction site a modified
approach of Sergeant and Mikaelian [89] was used (described in Wu et al. [90]). (Figure 9)
Primers pBSK_for, pBSK_rev and pBSK_SapI_mut were used in this experiment
(Figure 8).
29
Figure 8. A scheme of SapI site containing region and primers used for site-directed mutagenesis with their
annealing sites indicated. pBSK_for and pBSK_rev are flanking primers, and pBSK_mut is a mutagenic primers.
Restriction sites SacI and XceI used for further subcloning of amplified product are also depicted, along with
SapI restriction site.
Reannealing temperature and temperature at which the pBSK_for and pBSK_rev
primers do not bind to the plasmid DNA were determined practically using gradient PCR.
Further, the mutagenesis was performed in a single PCR reaction using all three primers and
pBSK vector with following PCR program:
1. 94 °C - 1 min
2. 94 °C - 45 sec
3. 72 °C (-1 °C per cycle) - 1 min
4. 72 °C - 1 min
5. GOTO 2 REP 10
6. 94 °C - 45 sec
7. 62 °C - 1 min
8. 72 °C - 1 min
9. GOTO 6 REP 6
10. 94 °C - 45 sec
11. 80 °C (Tra) - 45 sec
12. 42 or 52 °C - 1 min
13. 72 °C - 1 min
14. GOTO 11 REP 3
15. 72 °C - 2 min
16. GOTO 6 REP 6
17. HOLD AT 4 °C
The resulting PCR product of correct size was purified from gel, digested with SacI +
XceI restriction enzymes and ligated to a pBSK digested with the same enzymes, producing
plasmid pBSKmut. Incorporation of the mutation was controlled by SapI digestion.
Reaction mix (100 µl):
10x Phu buffer - 10 µl
2 mM dNTP mix - 10 µl
pBSK_for - 6 µl (60 pmol)
pBSK_rev - 6 µl (60 pmol)
pBSK_SapI_mut - 0.8 µl (8 pmol)
Template DNA - 1.2 µl (120 ng)
Pfu polymerase - 1 µl (2 U)
H2O - 65 µl
30
Figure 9. A schematic drawing of the mutagenesis process. During the first step, a single stranded mutagenic
DNA (mtDNA) is amplified from a plasmid with high Tm mutagenic primer and with high annealing
temperature at which flanking primers cannot anneal. During the second step, the megaprimer is produced with
one of the flanking primers from the smDNA template at low annealing temperature and low denaturing
temperature, at which where the plasmid DNA is not denatured. By repeating the above described procedures
(step 3), the full-length mutated DNA is synthesized first with a megaprimer at high annealing temperature and
then a complementary strand is synthesized by another flanking primer at low denaturing and low annealing
temperatures. Redrawn and modified from Wu et al. (2005) [90]
2.2.5.2.
2.2.5.2.
2.2.5.2.
2.2.5.2.
2.2.5.2.
2.2.5.2.
2.2.5.2.
2.2.5.2. Restriction-free cloning.
A restriction-free cloning method described by Ent and Löwe (2006). was used in this
work for construction of several plasmids such as SapI site containing vectors, F2A peptide
containing vectors and chimeric pCMV42 7G8H vector. This is a two-step PCR-based
approach, which involves amplification of the desired insert with specific primers in such
way, that the ends of the resulting fragment are complementary to the host vector at the
desired site of insertion. The amplification product serves further as a pair of primers in the
linear amplification reaction around a circular plasmid, thus eliminating the need for
restriction endonuclease recognition sequences (Figure 10).
31
Figure 10. Schematic representation of restriction-free cloning process. 1. The fragment to be inserted is first
amplified by a pair of primers, which are partially complementary to the unique insertion sites of the host vector
(red and blue fragments), isolated from dam+ strain. 2. The resulting PCR product serves further as a pair of
primerrs in a linear amplification reaction around a circular plasmid. Once annealed to the vector DNA
polymerase (Phusion in our case) extends and incorporates the gene into a nicked circular molecule (dashed
lines). After the digestion of parental vector with DpnI, the remaining circular double-nicked and double-
stranded DNA is transformed to an appropriate host cell. Redrawn and modified from van den Ent and Löwe
(2005) [91].
At first, a fragment to be inserted was amplified with a pair of ~50 nt, HPLC-purified
primers. In such primers about a half of sequence is complementary to desired insert and
another half is analogous to a host vector sequence upstream or downstream from the site of
insertion. Following the amplification, fragment of correct size was gel-purified and used as a
pair of primers in a linear amplification reaction. When the reaction was completed, buffer
32
was changed to water and the PCR products were digested with 20 U of DpnI for 2 h to
remove methylated parental DNA. Products of correct size were purified from agarose gel and
used directly for transformation. The correctness of insertion and absence of undesired
mutations possibly introduced during PCR was controlled by sequencing.
2.2.5.3. Design and construction of chimeric 7G8 mAb genes
All operations with antibody heavy and light chains were performed in an intermediate
vector – pBluescript II SK+ (pBSK). Human IgG1 heavy chain (together with the downsteam
intron sequence) and kappa light chain were cloned to MCS of pBSK vectors from pCMV42
vector using restriction enzymes XhoI + EcoRI for HC (~2500 bp) and XhoI + Cfr42I for LC
(~750 bp), generating plasmids pBSK42HC and pBSK42LC, respectively. Variable heavy
(VH) and variable light (VL) region coding sequences were changed in these vectors for those
of 7G8 mAb using RF-cloning method. Briefly, 7G8 VH and VL sequences were amplified
from pJET 1.2 vectors using primers HC_7G8_up_site_ins + HC_7G8_down_site_ins for
HC, and LC_7G8_up_site_ins + LC_7G8_down_site_ins for LC (Table 1). The resulting
products were purified from gel and used as a pair of primers (or, a „megaprimer“) in linear
amplification reactions using respective pBSK plasmids as templates (pBSK42HC for heavy
chain and pBSK42LC for light chain). After 2 h DpnI digestion the PCR products were
transformed to E.coli DH5α cells. Purified plasmids were controlled by sequencing. For
eucaryotic mAb expression, modified heavy and light chains were transferred back to
pCMV42 vector using enzymes Eco72I + BshTI for HC and Bsp119I + Cfr42I for LC,
producing a plasmid named pCMV_7G8H.
2.2.5.4. Design and construction of SapI vector system.
Sequences, coding for human HC and LC were transferred to pBSKmut from
pCMV42 just as described previously, generating plasmids pBSKmutHC42 and
pBSKmutLC42, respectively.
Sequences coding for murine IgG1 and IgG2a heavy chains were transferred to
pBSKmut from vectors pCMV65 (IgG1) and pCMV83 (IgG2a). Plasmids were digested with
SacI enzyme and corresponding fragments were gel-purified and ligated to pBSKmut digested
with the same enzyme, generating plasmids pBSKmutHC65 and pBSKmutHC83.
33
Sequence coding for murine light chain was transferred to pBSKmut in the same way
as the human light chain coding sequence, generating plasmid pBSKmut_mLC. Light chain
coding sequence of pCMV83 vector was identical to that of pCMV65 vector. Presence and
correct orientation of insert were confirmed with restriction digest (RD) analysis.
SapI restriction sites were inserted into generated pBSKmut vectors (bearing whether
heavy or light antibody chains) using RF-cloning strategy. First, VH and VL regions were
amplified with SapI restriction site containing primers, that were also partly complementary to
host vectors. Further, these amplification products were used in a linear amplification reaction
to generate plasmids with desired sites. Two SapI restriction sites in opposite orientations
were introduced into each target plasmid. One at the junction between leader sequence and
VH or VL sequence (antisense orientation), and another (sense orientation) whether at a
junction between VH (or VL) and constant region, or inside of a constant region of heavy
chain (CH), few nucleotides downstream the VH/CH junction (only for pBSKmutHC65 and
pBSKmutHC 83 vectors). Generated constructs are listed in a Table 1. Generation of a panel
of recombinant mAb expression constructs using SapI vector system.
Variable heavy and variable light regions of mAbs 7G8 and 8B2 were amplified from
corresponding pJet 1.2 plasmids/hybridoma cDNA using primers containig SapI restriction
site sequences (Table 1). PCR products of correct size were purified and digested with SapI.
pBSKmutSapI vectors containing different heavy and light chains were also digested with
SapI enzyme and ligated together with corresponding SapI-digested PCR fragments. Ligation
products were then transformed into chemically competent DH5α cells. Absence of mutations
was verified by sequencing. Further, HC and LC with changed VH and VL regions were
trasnferred from pBSKmutSapI vectors to pCMV vectors for expression exactly as described
in previous sections. Generated expression constructs were named pCMV_7G8M,
pCMV_8B2M and pCMV_8B2H. For the expression of chimeric 7G8 previously generated
pCMV_7G8H vector was used. The „H“ letter stands for human constant regions and the „M“
letter – for murine constant regions.
2.2.5.5. Generation of F-2A bicistronic constructs
For generation of F-2A constructs pBSK vectors with previously cloned antibody
heavy and light chains (see “Design and construction of SapI vector system”.) were used as
HC and LC donors. Heavy chains were cut out from the pCMV vectors using enzymes
HindIII and SalI. Light chains were cut out with XhoI and Cfr42I. Considering that enzymes
34
SalI and XhoI leave the same overhangs after cleavage, both HC and LC could be then
simultaneously ligated to pBSK vectors cut with HindIII and Cfr42I. The linker containing
DNA coding for furin cleavage site, SGSG spacer and F-2A peptide was inserted between the
heavy and light chains using RF-cloning method. Then, the whole sequence coding for HC
and LC (with a short linker between them) was transferred from each pCMV vector to pBSK
vector, generating pBSKF2A plasmids (Table 1).
A DNA sequence coding for a furin cleavage site, SGSG linker and Foot-and-Mouth
Disease Virus (FMDV) 2-A peptide (F-2A) was kindly provided by Radi Tegova (Icosagen
Group). The sequence was amplified with specific primer pairs (Table 1) and purified from
2% agarose gels. Resulted fragments were fused to pBSKF2A vectors directly between heavy
and light chains using the RF-cloning method. The whole sequence – HC-F-2A-LC – was
transferred from each pBSK vector to pCMV vectors for expression using Eco72I and Cfr42I
enzymes (generated plasmids – pCMVF2A, Table 1). All constructs generated by PCR were
analyzed by sequencing.
2.2.5.6. Grafting of 7G8M FR1 regions to 7G8H antibody
The VH and VL regions were transferred from pCMV vector bearing the 7G8M
sequence to the pCMV vector containing 7G8H sequence. For transfer of the VH region
enzymes Eco72I and Bpu1102I were used. Resulted constructs were sequenced, and correct
plasmid was used for transfer of the 7G8M VL region. Both, donor and host vectors were
digested with BpiI, which cleaves given plasmids at three sites, leaving different sticky ends
at each cleavage site. The fragment, containing the desired sequence of 7G8M was then re-
ligated with two remaining fragments obtained from the pCMV_7G8H digestion. Generated
constructs were analyzed by sequencing and clones bearing the correct plasmid were chosen
for expression.
2.2.6. Production and purification of recombinant mAbs
The novel technology, called QMCF Technology, developed by Icosagen Group
(Estonia) was utilized in this study for the large-scale production of murine- and chimeric
recombinant monoclonal antibodies. In particular, secretable recombinant mAbs were
produced by mammalian cells, based on chinese hamster ovary (CHO) and purified by protein
G sepharose (GE Healthcare) affinity chromatography at Icosagen facility.
35
Table 1. Constructs generated during this study (primer sequences are available upon request).
F- forward R- reverse
Primers (sequences are available
upon request) Method
Generated constructs (intermediate)
Expression constructs
mAb/ Comments
F ----> VH(for)
Ligation mAb VH regions: pJET_8B2_VH pJET_7G8_VH
R <---- VH(back) or VH_rev_IgG1, or VH_rev_IgG2a
F ----> VLκ(for) mAb VL regions: pJET_8B2_VL pJET_7G8_VL
R <---- VLκ(back) or VK_rev_CLk
F ----> HC_7G8_up_site_ins
RF-Cloning
pBSK_HC_7G8H
--> pCMV42_7G8H 7G8H R <---- HC_7G8_down_site_ins
F ----> LC_7G8_up_site_ins pBSK_LC_7G8H
R <---- LC_7G8_down_site_ins
F ----> HC65ForSapI
RF-cloning
pBSKmut_HC65_SapI_FR
HC65 - mouse IgG1 heavy chain
R <---- HC65RevFRSapI
F ----> HC65ForSapI pBSKmut_HC65_SapI_ Const
R <---- Hc65RevIgG1SapI
F ----> HC83ForSapI pBSKmut_HC83_SapI_FR
HC83 - mouse IgG2a heavy chain
R <---- HC83RevFRSapI
F ----> HC83ForSapI pBSKmut_HC83_SapI_ Const
R <---- HC83RevIgG2aSapI
F ----> HC70ForSapI
pBSKmut_HC42_SapI
HC42 - human IgG1 heavy chain
R <---- HC70RevFRSapI
F ----> LCAllForSapI
pBSKmut_mLC_SapI
mLC - mouse kappa light chain
R <---- LC6583RevSapI
F ----> LCAllForSapI pBSKmut_hLC_SapI
hLC - human light chain
R <---- LC70RevSapI
F ----> 8B2VH6570ForSapI Ligation pBSKmut_HC42_8B2VH
--> pCMV42_8B2H 8B2H R <---- VHAllRevFRSapI
F ----> 8B2VLForSapI Ligation pBSKmut_hLC_8B2VL
R <---- VL70RevSapI
F ----> 8B2VH83ForSapI Ligation pBSKmut_HC83_8B2VH_FR
--> pCMV83_8B2M_ FR
8B2M
R <---- VHAllRevFRSapI
F ----> VLAllForSapI Ligation pBSKmut_mLC_8B2VL
R <---- VHAllRevFRSapI
--> pCMV83_8B2M_ Const
F ----> 8B2VH83ForSapI Ligation pBSKmut_HC83_8B2VH_const
R <---- VH83RevIgG2aSapI
F ----> VH6570ForIgG1SapI Ligation pBSKmut_HC65_7G8VH
--> pCMV65_7G8M 7G8M R <---- VH65RevIgG1SapI
F ----> VLAllForSapI Ligation pBSKmut_mLC_7G8VL
R <---- VL6583RevSapI
F ----> F2A_hIgG1_fw RF-cloning
pBSK_8B2H_F2A and pBSK_7G8H_F2A
pCMV_8B2H_F2A and pCMV_7G8H_F2A
8B2H_F2A
R <---- F2A_rev_all 7G8H_F2A
F ----> F2A_mIgG2a_fw RF-cloning
pBSK_8B2M_F2A
pCMV_8B2M_F2A 8B2M_F2A R <---- F2A_rev_all
F ----> F2A_mIgG1_fw RF-cloning
pBSK_7G8M_F2A
pCMV_7G8M_F2A 7G8M_F2A R <---- F2A_rev_all
36
2.2.7. HCV RdRp purification
HCV RdRp was purified using the original protocol of Binder et al, (2007) [92] with
minor modifications made to meet the conditions of our lab. Plasmid pET-19b was
transformed to chemically competent E. coli BL-21 (DE-3) cells. Bacterial cells were grown
in LB medium at 37°C. When the optical density at 600 nm reached 0.7-0.8, NS5B expression
was induced by adding Isopropyl-β-D-Thiogalactoside (IPTG) to a final concentration of 0.6
mM and bacterial culture was shifted to a room temperature. After 8 h of induction, cells were
pelleted by centrifugation at 6000×g for 10 min, washed once with 1xPBS and resuspended in
40 ml of Lysis Buffer I (LBI: 100 mM Tris-HCl [pH 8.0], 100 mM NaCl, 1mM MgCl2, 2%
Triton X-100, 2 mg/ml lysozyme (Amresco), 3 kU of DNase (Calbiochem), and protein
inhibition cocktail (Roche)). After a 30 min incubation at +4 °C lysate was centrifuged at
20000×g for 10 min at +4 °C, supernatant was removed and the pellet was resuspended in 5-7
ml of Lysis Buffer II (LBII: 20 mM Tris-HCl [pH 7.5], 500 mM NaCl, 2% Triton X-100, 10
mM imidazole, 30% glycerol, 10 mM 2-mercaptoethanol, protein inhibition cocktail
(Roche)). The suspension was further sonicated on ice in 1-ml aliquots 5 times for 20 seconds
at an output control setting of 6 using Bandelin Sonopuls HD2070 sonifier. After 30 min
centrifugation at 21,100×g at +4°C, the supernatant (cleared lysate) was mixed with 400 µl of
1:1 Ni-nitriloacetic acid (Ni-NTA) agarose slurry (QIAGEN), incubated for 1 h at a rotary
shaker at +4 °C and centrifuged for 5 min at 500×g. Supernatant was discarded and Ni-NTA
matrix with bound NS5B was packed onto the column (Bio-Rad), washed 4 times with 4 ml
of LB II containing 50 mM imidazole and then eluted with 1 ml (5×200 µl fractions) of LB II
containing 250 mM imidazole. Eluate was stored in small aliquots at -80°C. Fractions were
analysed on 12% SDS-PAGE and protein concentration was determined in Bradford protein
assay (Bio-Rad) using BSA standart.
2.2.8. Protein analysis and quantification methods
2.2.8.1. Protein quantification using Bradford protein assay
Protein quantification was performed by a modified Bradford method using Bio-Rad
protein assay dye. 5 µl of BSA dilutions (for generation of a standart curve) and NS5B
samples were mixed with 800 µl of H2O in 1.5 ml tubes, then 200 µl of the Bio-Rad protein
assay dye (pre-warmed to room temperature) was added to each tube. Reactions were
37
incubated for 20 min at room temperature and OD was read at 595 nm using a Heλios
(Thermo) spectrophotometer. Protein concentration were calculated from the standard curve
equation.
2.2.8.2. Western blot
NS5B samples, containing known amount of protein were mixed with the same
volume of 2x Laemmli buffer [93] and denatured for 10 min at 95°C prior to SDS-PAGE.
After resolving the samples on 12% polyacrilamide gel, proteins were transferred to a PVDF-
membrane (Millipore) using a semi-dry transfer method in a Trans-Blot SD apparatus (Bio-
Rad). Further, membrane was incubated for 1-2 hours in a blocking solution I (BSI: 5% w/v
Non-fat dry milk (NFDM), 0.1% Tween-20, 1xPBS) and then incubated with a primary mAb
specific to the NS5B in a blocking solution II (BSII: 2% NFDM, 0.1% Tween-20, 1xPBS).
After that, the membrane was washed 3 times for 15 min with a washing solution (50 mM
Tris-HCl pH 7.5; 150 mM NaCl; 0.1% Tween-20) and incubated for 1 h with a secondary
mAb (anti-mouse or anti-human HRP-conjugated IgG) in the blocking solution II. After 3x15
min washings, NS5B specific signals were developed using ECL-Plus kit (Amersham
Pharmacia Biotech) and detected by exposing the membrane to a roentgen film (Fuji).
2.2.9. Enzyme-linked Immunosorbent Assay (ELISA)
ELISA was performed according to Pfaff et al. [94]:
96-well microtiter plates (Nunc) were coated with peptides (5 µg/µl) or full-length
NS5B (0.25 µg/µl) and incubated overnight at 37 °C. After incubation, wells were washed
twice with 200 µl of 1xPBS and saturated with 1% BSA in PBS (30 min at 37 °C). 50 µl of
mAb dilutions (or hybridoma supernatant dilutions – 1:10 to 1:100000) from 0.1 pg/µl to 10
ng/µl were placed in the wells and incubated for 1 h at 37 °C. After washing 5 times with 200
µl of PBS/Tween-20 (0.05%), samples were incubated for another 1 h with 100 µl of
horseradish peroxidase (HRP) conjugated rabbit anti-mouse (at a ratio of 1:10000), or goat
anti-human IgG (at a ratio of 1:8000) in 1% BSA/PBS. After several washings with
PBS/Tween-20 (0.05%) wells received 100 µl of a substrate solution (Bio-Rad) containing
3,3’,5,5’-tetramethylbenzidine in dimethylformamide and hydrogen peroxide at a ratio of
10:1. After development of deep blue color, reactions were stopped with 1N H2SO4 and OD
was read at 450 nm with a “Sunrise” (Tecan) multiscan photometer.
38
2.2.10. Changing mAb buffer by ultrafiltration
For RdRp assays mAb buffer was changed to TN (10 mM Tris; 100 mM NaCl) using
Amicon Ultra-4 Centrifugal Filter Units with Ultracel-10 membrane (Millipore). First, units
were equilibrated by centrifugation with 4 ml of TN for 30 min at 4000×g at room
temperature in a swinging-bucket type rotor (Sigma). Then, flowthrough was discarded and
mAb solution (~200 µg mAb) diluted with TN to a total volume of 4 ml was added. Units
were centrifuged for 12-15 min at 4000×g at room temperature to get residual volume of
approximately 100 µl. Then, another 4 ml of TN were added and centrifuged. The procedure
was repeated 4 times and residual solution, containing concentrated antibody, was transferred
to a new 1.5 ml tube. After buffer exchange mAbs were stored at +4°C. Concentrations of
mAbs were determined with a ND-1000 spectrophotometer at 280 nm using an IgG antibody
extinction coefficient of 1.37. Alternatively, concentration was measured using Bradford
protein assay (Bio-Rad) with IgG standard (Pierce).
2.2.11. Primer-dependent RdRp assays
RdRp assays were performed essentially as described by Nikonov et al. [87]
For all RdRp assays mAb buffer was changed to TN (as described above). Poly (rC)
template was mixed with oligo (rG)12 primer, denatured for 2 min at 95°C, and incubated for
5 min on ice before use.
Primer-dependent RdRp assays were performed in 1xRP buffer (10 mM Tris-HCl, pH
7.5; 12.5 mM KCl; 2.5 mM MgCl2; 0.5 mM DTT; 0.5 mM EGTA, pH 7.5) and contained
~0.12 µmol of HCV NS5B polymerase, various mAb dilutions (0.6 – 1.2 µM), 0.4 µg of
poly(rC) pre-annealed to 4 pmol of oligo(rG)12 primer, 10 units of RiboLock Rnase Inhibitor
(Fermentas), NTP – CTP, ATP and UTP at a final concentration of 0.5 mM and 1 µCi of [α-
32P]-GTP per reaction. First, NS5B and mAbs were preincubated at +4°C for 10 min, then
pre-annealed primer-template mix was added and incubation was prolonged for another 10
min. After that, reactions were initiated by addition of NTP (CTP, ATP, UTP and [α-32
P]-
GTP). The reactions were incubated for 1 h at 25°C and then stopped by addition of 1 ml of
ice-cold 10% trichloroacetic acid (TCA), 0.5% tetrasodium pyrophosphate (Na4PPi) and 100
µg of herring sperm DNA. After 30 min incubation on ice, samples were filtered through
glass-microfiber GF/C filters (Whatmann and Schelicher & Schuell) and washed 2 times with
5 ml of 1% TCA/0.1% Na4PPi solution. After that, filters were placed to scintillation vials and
39
dried for 15 min at 65 °C. The activity of NS5B polymerase was then measured by liquid
scintillation counting of [32
P]-GMP incorporated into the synthesized RNA (bound to the
GF/C filters) using ScintiSafe 3 liquid scintillation cocktail (Fisher Scientific) and
WinSpectral counter (Wallac). The primer-dependent assay for wild type and mutant (GND)
NS5B polymerases was performed in the same manner, but with different concentrations of
the enzyme and without mAbs (thus, the first 10-min preincubation step was omitted).
40
3. Results and discussion
3.1.1. Cloning of antibody variable regions
Two mouse hybridoma cell lines 7G8.1 and 8B2.1 producing monoclonal antibodies
(mAbs) 7G8 (IgG1) and 8B2 (IgG2a) were generated previously in our lab. 8B2 and 7G8
specifically inhibited RdRp activity of HCV NS5B polymerase [87]. We decided to clone
variable regions of these mAbs. For this purpose, genomic DNA extracted from hybridoma
cell line producing specific mAb could not be used, because it contained non-transcribed
DNA encoding nonspecific antibody fragments. That is why hybridomas total RNA was used
to isolate antibody sequences corresponding to variable regions of 7G8 and 8B2.
Subsequently, total RNA was used for reverse transcription-polymerase chain reaction (RT-
PCR) as described in “methods” section.
Total RNA was extracted from hybridomas with TRIzol reagent and its integrity was
analyzed on agarose denaturing gel. The preparations had two sharp bands, corresponding to
28S and 18S rRNA, and the 28S band is approximately as twice intence as 18S band. Taken
together with the absence of a low molecular weight smear (below 5S), these data indicated
that the total RNA was intact.
Figure 11. Denaturing formaldehyde RNA gel-electrophoresis. ~1 µg samples of total hybridoma RNA were run
on 1% denaturing agarose gel. The 28S and 18S bands (indicated by arrows) are sharp, with 28S band being
approximately twice as intense as 18S. The absence of smear below 5S band demonstrates that there is no
degraded low molecular weight RNA.
For amplification of antibody heavy and light chain variable domains degenerate 5’
primers designed by Wang et al. were used (Table 1) [95]. The design of these primers is
41
based on a relatively conserved sequence in the framework one (FR1) regions. The primers
have degeneracies at 7 positions in order to cover the majority of sequence combinations at
these regions. The rest of these primers is complementary to the most conserved nucleotides
in FR1. Two different types of 3’ primers were used: first – primers, complementary to the
framework 4 (FR4) regions, that have a high degree of conservation (design was based on
Orlandi et al., [96]). Second type – primers, complementary to isotype-specific constant
region sequences [95], (see Figure 12).
Figure 12. Schematic representation of antibody variable region with primer annealing sites. Framework regions
are labelled as FR and complementarity-determining regions as CDR with numbers indicating the order of these
regions. L – leader peptide sequence. Primer binding positions are shown as arrows.
As seen from Figure 13A, majority of mAb variable regions could be succesfully
amplified with primers, complementary to framework regions. Amplification of 8B2 VH
region, however, was less efficient, than in case of the other mAbs. This problem was solved
by utilizing the 3’ primer, complementary to mouse IgG2a constant chain (Figure 13B). As
further sequence analysis showed, two amino acids in FR4 of 8B2 VH region were different
from the most frequent ones (on which the design of the initially used 3’ primer was based).
This explained why this region could not be amplified with the proofreading PhusionTM
DNA
polymerase.
42
Figure 13. Amplified antibody variable regions. A) VH and VL regions of mAbs 8B2, 7G8 and 10D6 (control),
amplified with Taq DNA polymerase using degenerate forward primers and reverse primers complementary to
framework regions. Specific bands corresponding to the variable regions are shown with arrow. B) VH region of
8B2 mAb, amplified with PhusionTM DNA polymerase, with the help of reverse primer complementary to
IgG2a constant chain. The amplified fragment is ~90 nt longer then those on Figure 13A, because it contains a
part of the constant region sequence.
Control reaction without reverse transcriptase, without RNA template and without
both enzyme and RNA (Figure 13) did not yield any amplification products, indicating that
there was no DNA contamination during RT-PCR. Subsequently, amplified fragments were
inserted into pJet 1.2/blunt vector for sequencing generating following constructs:
pJet7G8VH, pJet7G8VL, pJet8B2VH, pJet8B2VL. Multiple sequence alignment and analysis
allowed to identify clones with the consensus sequence – those, not containing any undesired
mutations that could be possibly introduced by PCR. Clones containing the consensus
sequences for given antibodies were chosen for further manipulations.
3.1.2. Construction of chimeric 7G8 antibody genes
Antibodies, capable of inhibiting key enzymes in HCV replication, are clearly of great
interest, because of their possible use in anti-HCV therapy. However, murine mAbs, if
administered to human, are effectively recognized by the host immune system as foreign
43
antigens, which leads to rapid clearance of these mAbs from the individual’s organism. There
are some approaches, first described and applied more than 20 years ago, that can help to
eliminate the problems associated with this defense mechanism. For example, murine
antibodies can be chimerized or humanized (reshaped). Humanization is described in details
in the theoretical part of this thesis. The term „chimeric“, when applied to an antibody, means
that the variable regions of this antibody are from one organism (for example, mouse), and all
other regions are from host organism (for example from human) (Figure 6). Such antibody
would contain only ~25% of original murine antibody sequence, what could significantly
lessen the chance of being recognized by a human immune system. There are many examples
of antibodies that were successfully chimerized or humanized, and currently used in antiviral
or anti-cancer therapy (see „background“ part). Though, humanization can make antibody
even more human-like, than chimerization, it is much more difficult process. That is why we
pursued the construction of chimeric antibodies first, to prove that our mAbs can function in
this format, and that all the mAb gene cloning, engineering and expression technology works
in our hands.
Initially the approach was applied only to one mAb (7G8) in order to get a „proof of
principle“ that the whole system for creation of recombinant antibodies in such way works.
Due to the use of the degenerated primers we were unable to determine the native sequence
for the 1st, 3
rd, 5
th and 6
th amino acids of VH, and 4
th, 7
th and 8
th amino acids of VL regions of
our mAbs. Thus, during this pilot experiment of 7G8 chimerization we decided to change
these amino acids for those of a published humanized anti-RSV mAb [63], because the fact
that this mAb maintained its biologic activity after humanization seemed to increase our
chances of success.
Expression vector pCMV_7G8H, containing 7G8 mAb variable region sequences
inserted between the leader peptide coding fragment (needed to direct the antibody to ER for
packing and secretion) and the constant region sequence of corresponding chains, was
constructed as described in „Methods“ section (Figure 18). This chimeric 7G8 antibody was
analyzed in ELISA and Western blot assays for histidine-tagged bacterially expressed NS5B-
binding ability. As could be seen from Figure 14, chimeric 7G8 reacted strongly in Western
blot assay, whereas its activity in ELISA was relatively weak (what later turned out to be the
issue of the secondary antibody used in this experiment (data not shown)). These experiments
demonstrate that the recombinant antibody generated using the described approach
maintained its affinity to the antigen. So, it was decided to generate the high-throughput
44
vector system for generation of similar recombinant constructs not just for 7G8 and 8B2
mAbs, but for virtually any murine antibody of either IgG1 or IgG2a isotype.
Figure 14. Chimeric 7G8 activity. A) A photo of ELISA test results for chimeric 7G8 (7G8H) ability to
recognize NS5B RNA polymerase in native conformation. The supernatant dilutions of a cell culture transfected
with 7G8H expressing plasmid were used in this experiment. Supernatant from a non-transfected culture and an
anti-BPV-1 E2 region antibody were used as negative controls (shown with arrows). B) Western blot with 7G8H
and NS5B polymerase. Amount of input NS5B is shown at the top.
3.1.3. Generation of a vector system for high-throughput construction of
recombinant antibody genes.
Although, the RF-cloning method is very good for generating difficult constructs, it
cannot be used in a high-throughput manner, because of the need of expensive high-quality
primers and also because all constructs generated by PCR need additional sequence control.
Considering these facts, there clearly was a need for a system, which would allow easy and
rapid generation of recombinant antibody genes. One of the simpliest and most wide-spread
techniques in molecular cloning is the use of restriction enzymes, so the RD-based approach
was developed in collaboration with Icosagen Group (Estonia) and used for the construction
of such system. For that purpose, a restriction enzyme named SapI (or LguI) was chosen. SapI
recognition site is 7 bp long, which makes it more rare, compared to typically occuring 6 and
4 bp long restriction sites. The site is also non-palindromic and the cleavage occurs
downstream of the recognition sequence, leaving sticky ends, which usually differ from each
other, preventing the re-circularization of the digested sequence and allowing the ligation of
45
the gene of interest in correct orientation (see Figure 15). Due to the fact, that the cleavage
site differs from the recognition sequence, the restriction sites can be removed after digestion.
Thus, products of the subsequent ligation do not contain any foreign sequences.
Figure 15. SapI recognition site and digestion specifics. SapI recognition sequence is drawn in red in both direct
and reverse orientations. Adjacent nucleotides designated with N, meaning any of four dNTPs. The scissors
icons and a broken line depict the way DNA is cleaved by SapI. In the lower row, SapI cleavage products with
protruding 5’ ends are shown.
Utilizing the RF-cloning method, SapI restriction sites were introduced to pBSK
vectors with following pre-inserted antibody chains: human light and heavy (IgG1 isotype)
chains, murine IgG1 and IgG2a heavy chains, and murine kappa light chain (Table 1). The
naturally occurring SapI cleavage site of the plasmid was eliminated by site-directed
mutagenesis (see “methods“). The 5’ SapI site was inserted in reverse orientarion in such way
that cleavage site located right at the junction between signal peptide and VH (or VL) region.
The 3’-end SapI restriction site was inserted in direct orientation so that it located at the
junction between the VH (or VL) region and the constant region of corresponding chain. For
mouse IgG1 and IgG2a additional constructs were generated. These constructs differed from
the previously described in a way that 3’-end SapI sites were introduced into their constant
regions, in order to allow the ligation of VH fragments amplified with the help of the
VH_rev_IgG1 and VH_rev_IgG2a primers (Table 1, Figure 12, Figure 16). All generated
constructs are schematically drawn in Figure 16.
46
Figure 16. Schematic representation of generated SapI restriction sites containing vectors. The positions of the
SapI sites are shown as blue pentagons, where the tip of each pentagon determines the orientation of the
restriction site (also indicated by arrows at the top of the scheme). L – leader sequence, VH – variable heavy
region, VL – variable light region (kappa), CH – constant heavy region, CL – constant light region. pBSK HC or
LC – pBluescript II SK(+) plasmids containing respective HC or LC genes.
Next, primers containing the SapI restriction sites were designed on a basis of the
primers used for initial cloning of antibody variable domains. The VH and VL regions
amplified with these primers would contain the SapI site in the correct orientation. Also, the
sticky ends, left after digestion with SapI restrictase would be exactly complementary to those
of respective vectors (digested in the same way). At large, utilization of such vector-primer
system allows to amplify variable region from virtually every mouse hybridoma and join them
directly to constant regions of human origin. This system might be further customized to
allow the creation of mAb chimeras of any two or more organisms. Because given project is
47
therapy oriented, only human Ab chains were used as foreign constant region donors. All
generated plasmids were controlled by sequencing for the presence of the desired insertion.
The principle of the SapI system is shown schematically on Figure 17.
Figure 17. Scheme, representing the process of generation of recombinant antibody genes using the SapI vector-
primer system. Variable region amplified from hybridoma cDNA shown in green, host variable region – in gold.
Scheme of amplification of variable regions from hybridoma cDNA (with primer pairing sites) is drawn in a
dashed box. SapI site-containing primers are shown as arrows – green part is complementary to the FR regions
of variable domain and blue part – is a SapI site-containing sequence. SapI restriction sites in PCR products and
in host vector are shown as blue pentagons, where tip of each pentagon indicates the orientation of the
corresponding site. Scissors icons indicate where the cleavage occurs. L – leader peptide.
48
3.1.4. Construction of a panel of recombinant 7G8 and 8B2 monoclonal
antibodies.
Utilizing the above described system, a panel of recombinant 7G8 and 8B2 mAbs was
generated, which included chimeric mouse/human (containing mouse variable regions and
human constant regions) antibodies and their mouse/mouse (containing mouse variable
regions fused to codon-optimized mouse constant chains) analogues (Figure 18A). All
variable regions could be efficiently amplified with Phusion DNA polymerase, using SapI
containing primers. All fragments and vectors containing SapI restriction site were
successfully digested and ligated in correct orientation without the requirement of any
optimization. Recombinant mouse mAbs were constructed in order to compare their biologic
activity with chimeric and hybridoma mAbs biologic activities. Two variants of mouse 8B2
antibody were constructed. First, VH region was amplified with the help of 3’-end primer
complementary to FR4 region. Second, the same region was created by using 3’-end primer
complementary to IgG2a constant region (Table 1). These constructs differed by two amino
acids in FR4 regions and were designated respectively 8B2MF and 8B2Mconst. As the
subsequent experiments did not reveal any difference in their function (data not shown), only
one of them, 8B2MF (further referred to as 8B2M), was used for further experiments.
Chimeric 8B2 was designated as 8B2H and 7G8 chimeric and murine mAbs as 7G8H and
7G8M, respectively. The recombinant mAb genes were cloned to pCMV vectors for
expression purposes. The expression vectors, containing the above described antibody genes
are listed in Table 1.
49
Figure 18. Schematic representation of recombinant antibody genes, generated in this study. A) Recombinant
genes for two-promoter expression and B) “bicistronic” F2A recombinant constructs for single-promoter
expression. L – leader sequence, VH and VL – variable heavy and light regions, CH and CL constant heavy and
light regions, 2A – Foot-and-Mouse Disease Virus 2A peptide encoding sequence. Orange and green boxes are
sequences coding for furin cleavage site and SGSG spacer, respectively. Schematic drawings of expression
vectors with the relative positions of gene expression cassettes are shown under the corresponding gene schemes.
3.1.5. Design and construction of bicistronic mAb expression vectors
utilizing the F-2A peptide sequence.
As pCMV expression vectors utilize two promoters, separately for mAb heavy and
light chains, the molar ratio of the expressed chains is hard to control and with high
probability is not equal, due to a competition between promoters. In this case, efficiency of
production of functional antibody dimers is limited by the efficiency of transcription from the
50
less successful promoter. Besides affecting the total yield of functional antibody, excessory
immunoglobulin chains may also form aggregates and cause precipitation of other proteins,
what can adversely impact the therapeutic potential of the antibody. Thus, for optimal
production and use ratio of expressed antibody chains should be 1:1.
In order to overcome this two-promoter issue, we decided to construct so called
bicistronic mAb expression vectors, according to Fang et al. [97], in which both heavy and
light chain are expressed as a single open reading frame. In such vector, the 5’ end of the gene
encoding the light chain is connected with the 3’ end of the heavy chain through a sequence
directing the synthesis of a short peptipe linker (Figure 19). This linker consists of three parts.
First part is represented by the cleavage site of furin, – a serine protease normally presented in
animal cells [98-99]. Second part is a flexible four amino acid SGSG spacer, shown to
improve the furin cleavage rate [100]. And the last part is the 24 amino acid 2A peptide of the
Foot-and-Mouth Disease Virus (FMDV) (Figure 19). This type of a peptide was first
identified among picornaviruses, in the aphtoviruses sub-group, a typical example of which is
FMDV [101-102]. In these viruses multiple proteins are derived from a large polyprotein
encoded by a single open reading frame. 2A peptides contain the consensus motif at the C-
terminus (Asp-Val/Ile-Glu-X-Asn-Pro-Gly-Pro). The unique feature of this peptide is that
during translation the synthesis of peptide bond between glycine and the last proline residues
does not occur, resulting in the ribosome skipping to the next codon and cleavage of the
nascent protein between Gly and Pro (Donnelly et al., 2001).
Figure 19. Scheme of a typical “bicistronic” antibody gene for expression from a single promoter. Sequence
encoding furin cleavage site, SGSG spacer and FMDV 2A peptide is shown between the dashed lines. Scissors
indicate the sites where the polypeptide chain break occurs. L – leader peptide.
In the case of bicistronic mAb expression constructs, 2A peptide mediates separation
of light chain from heavy chain, leaving the proline residue fused to the N-terminus of the
51
light chain. The remaining part of the peptide linker is then cleaved from the C-terminus of
the heavy chain by cellular furin protease (Figure 19, 20). However, two additional amino
acids derived from the furin cleavage site (Arg and Ala) remain fused to the C-terminal end of
the heavy chain (Figure 20). Fortunately, these additional amino acids seem to have no effect
on the mAb functionality, at least as reported by Fang et al, [97]. Using this approach both
chains constituting mAb could be produced at equimolar ratio.
Figure 20. Scheme of the polypeptide processing during translation of a transcribed bicistronic antibody gene,
and biosynthesis of a corresponding antibody. Furin cleavage site and amino acids that remain fused to the
antibody after assembly are drawn in orange. The course of the expression and processing is indicated at the
right by arrows. Modified from Fang et al. (2005) [97]
The constructs containing the above described sequences (Figure 18B) were generated
on the basis of the pCMV expression vectors, as described in “methods”. The stop codon at
the 3’ end of the heavy chain coding sequence was eliminated during the cloning. The
sequences of the whole resulting ORFs were controlled by sequencing and transferred to the
pCMV plasmids, generating constructs pCMV7G8H-F2A, pCMV8B2H-F2A, pCMV7G8M-
F2A and pCMV8B2M-F2A. A scheme of typical F2A expression construct is presented in
Figure 19.
52
3.1.6. Expression and purification of enzymatically active NS5B
polymerase.
pET19b vector containing the NS5B gene under the control of bacteriophage T7
promoter (kindly provided by Andrei Nikonov) was used for wild type NS5B (wtNS5B) and
its inactive mutant (GND) overexpression in E.coli BL21 (DE3) strain. The resulting protein
had a molecular weight of approximately 66 kDa and contained a decahistidine tag fused to its
N-terminal end. The growth and purification of wtNS5B (or GND) was performed according
to Binder et al. [92], with minor modifications. The expression of the protein was confirmed
by SDS-PAGE, using non-induced cultures as a control (Figure 21A). Samples of expression
culture were taken at different time points – 4, 9, 12 and 18 hours after induction and analyzed
by SDS-PAGE. Results of this analysis (shown on figure 21A) indicate that the optimal time
for expression of NS5B at room temperature is between 4 and 9 hours, and after this time
amount of the product starts to decrease. Therefore, all expression cultures were further
induced for ~8 hours. Following the purification procedure, the eluate, washing and
flowthrough fractions were analyzed by SDS-PAGE (Figure21B). As it is seen on the Figure
21B, the protein was purified to near homogeneity with the majority of the protein coming out
in 2-nd and 3-rd fractions (lanes F1-F5). The flowthrough (lane Ft) contains the major part of
unbound cellular proteins, whereis only trace amount of proteins is seen in the washing
fraction, demonstrating the specificity of the Ni-NTA matrix to His-tagged NS5B.
Figure 21. NS5B A) expression and B) purification. Shown are 12% SDS-polyacrilamide gels stained with
coomassie brilliant blue (R-250). Marker proteins sizes are indicated at the left (in kDa). A) samples of NS5B
expression culture taken at various time points after induction are analyzed by SDS-PAGE, M – marker, NI –
non-induced control. 4h, 9h, 12h and 18h – hours post induction. NS5B specific band is indicated with an arrow
at the right. B) Different fractions collected during the purification of NS5B are analyzed along with series of
BSA dilutions for empirical quantification of NS5B yield. The amounts of input BSA are indicated above the
correspondent lanes. Ft – flowthrough fraction of NS5B, W – washing fraction, F1 – F5 are elution fractions.
NS5B specific band is indicated with an arrow at the right.
53
Relatively high content of NS5B in the flowthrough fraction can be explained by
inefficient disruption of cell membranes during homogenisation. Due to a highly hydrophobic
region at the C-terminus, majority of NS5B was found to be membrane-associated. Thus,
incomplete disruption of cell membranes could result in large amount of the protein remained
membrane-associated, what, in its turn, could interfere with the binding to Ni-NTA matrix.
Generally, purification of NS5B from 1 litre culture yielded approximately 0.5 mg. Enzymatic
activity of purified NS5B was controlled in a primer-dependent RdRp assay, using liquid
scintillation counting of radioactively labelled GMP, incorporated into synthesized RNA
(Figure 22). As it is seen on a figure 22, NS5B activity grows linearly when the enzyme
amount is being increased up to 800 ng and reaches a plateau at higher concentrations,
achieving the upper limit of approximately 170000 cpm. On the other hand, when the same
amounts of the GND mutant were used, only residual radioactivity of ~5000-7000 cpm could
be detected. These results indicate, that the purified wtNS5B is enzymatically active and pure
enough to be used in biochemical assays at concentrations up to 800 ng per reaction (since
there are no signs of activity inhibition at these concentrations) for testing the biologic activity
of different mAbs.
Figure 22. NS5B enzymatic activity. The amount of specific radioactivity (in counts per minute) incorporated
into the synthesized RNA (Y-axis) is plotted against the amount of NS5B in reaction (X-axis). Activity of NS5B
mutant inactive form (GND) is shown as black triangles, wtNS5B – white rectangles.
3.1.7. Biologic activity of recombinant 7G8 and 8B2 mAbs
The recombinant antibodies could be efficiently produced in CHO cells, using the
Icosagen QMCF technology (described in “methods” section), yielding up to 2.5 mg of mAb
for constructs utilizing two promoters. Interestingly, the yield was better for chimeric
54
antibodies (1.5 – 2.5 mg) than for murine ones (less than 1 mg). This could be possibly
explained by a predominance of expression from promoter of the light chain coding gene.
Resulting lower concentration of expressed heavy chains (F(c) regions) could reduce the
efficiency of antibody purification by a protein G affinity chromatography. Nevertheless,
yields of the antibodies produced using the QMCF technology (Icosagen Group) are
comparable with those can be obtained from hybridomas.
Expression of antibodies from bicistronic constructs resulted in ~2-fold increase in
antibody yields, indicating the possible benefits of this single-cassette expression system.
However, it was shown, that in case of high expression level, the amount of cellular furin is
not sufficient to cleave all synthesized protein molecules. This indicates that additional furin
might be provided in order to improve cleavage efficiency. Thus, this approach still requires
additional optimization, before it can be routinely used.
3.1.7.1. Biologic activity of recombinant mAbs in antigen binding assay
(ELISA).
The ELISA tests were performed in order to show whether the ability of recombinant
7G8 and 8B2 mAbs to bind the antigen (NS5B) is comparable with that of the hybridoma-
produced mAbs. ELISA was preferred to western blot method, because in this assay the
antigen remains in its native conformation. Also, comparing to WB, ELISA is a quantitative
method, where the efficiency of mAb binding is estimated by directly measuring the intensity
of the final sample solution color. Results of these ELISA experiments with different
recombinant and hybridoma mAbs are shown in Figure 23.
Figure 23. ELISA assay with A) 8B2 and B) 7G8 hybridoma and recombinant mAbs. ELISA specific signal
intensity (OD450, Y-axis) is plotted against mAb concentration (X-axis). 8B2.1, 7G8.1 – hybridoma mAbs.
8B2H, 7G8H – chimeric mAbs. 8B2M, 7G8M – recombinant native murine mAbs.
55
As it can be judged from these results, for the 8B2 mAbs the signal could be detected
starting from mAb concentration of 0.1 ng/µl, becoming saturated at a 10 ng/ml
concentration. The recombinant 8B2H and 8B2M mAbs show the same pattern of binding
efficiency, whereas the hybridoma 8B2 (8B2.1) gives a weaker signal at a concentration
between 0.1 and 10 ng/µl (probably due to the use of the old mAb stock). In case of the 7G8
mAbs, the specific signal could be detected already at 0.01 ng/µl mAb concentration,
achieving saturation at 1 ng/µl. For all three 7G8 antibodies used in the experiment the
binding profiles were somewhat different. Strength of the signal (at a mAb concentration of 1
ng/µl) decreases in the following order: 7G8M > 7G8H > 7G8.1. A weaker signal for
hybridoma 7G8 can be explained (at least in part) by the usage of the old mAb stock, whereas
difference between the antigen-binding activities of 7G8H and 7G8M antibodies is of another
origin (will be discussed later). All-in-all, these data indicate that recombinant mAbs
constructed in this study are capable of binding the native NS5B with at least the same
efficiency as hybridoma mAbs. Moreover, the recombinant 7G8M mAb shows even stronger
binding pattern, than its chimeric and hybridoma analogues.
3.1.7.2. Biologic activity of recombinant mAbs in primer dependent RdRp
assay.
The standart RdRp assay is a continuous polymerisation reaction, which consists of
repetitive initiation and elongation cycles. NS5B is a processive enzyme, capable of initiating
the elongation polymerization reaction from pre-annealed template/primer RNA substrate.
Polycytidylic acid (poly(C)) with randomly pre-annealed guanosine 12-meric
oligonucleotides ((rG)12 primer) is usually used in the RdRp assay because this
template/primer hybrid is shown to be utilized by NS5B with the highest efficiency [103]. In
contrast to, for example, highly processive DNA polymerases, HCV NS5B RNA polymerase
has noticeably lower processivity. NS5B constantly dissociates from the template/primer
substrate and then re-binds in order to initiate a new elongation cycle. Different 7G8 and 8B2
recombinant antibodies along with hybridoma mAbs were examined in this assay for their
ability to inhibit the primer-dependent synthesis of RNA by NS5B in vitro. The results are
presented in Figure 24.
56
Figure 24. Primer-dependent RdRp assay A) with 8B2 and 7G8 recombinant and hybridoma mAbs and B) with
8B2 F2A and 7G8 F2A mAbs + hybridoma mAbs. The order of the reagent addition is indicated at the top of the
graphs. X-axis shows the molar ratio of mAbs to NB5B in reaction. Y-axis displays the amount of specific
radioactivity incorporated into synthesized RNA (in counts per minute). The means of at least two indipendent
experiments with standart deviations are indicated. 8B2.1, 7G8.1, 9A2.1, 6G5.1 – hybridoma mAbs. Antibodies
designated with “H” letter – chimeric mAbs, and with “M” letter – recombinant native murine mAbs.
Obtained data clearly indicate that 7G8 and 8B2 mAbs, both recombinant and
hybridoma-produced, inhibit the RNA synthesis by the NS5B polymerase in a dose-dependent
manner in this in vitro assay. All 8B2 antibodies demonstrate highly similar inhibition
patterns, similar to those described by Nikonov et al. [87]. These mAbs inhibit the NS5B
polymerase approximately 4-5 fold. Starting from more than 2-fold mAb molar excess over
NS5B, the residual activity of the polymerase is only about twice higher than the background
(determined as the average activity of the non-functional NS5B GND mutant (not shown on
the graph)). At the same time, a control 9A2.1 hybridoma mAb seems to have no
57
concentration-dependent inhibitory effect upon the NS5B. A slight decrease in NS5B activity
at higher concentrations of the control mAb could be explained by purely physical
interference with polymerase-substrate interaction.
A different picture can be observed in case of 7G8 mAbs. 7G8H and 7G8.1 do show
the same functional properties, very similar to those described by Nikonov et al. [87],
however, mAb 7G8M does not. This antibody demonstrates an enhanced NS5B inhibiting
activity, compared to all other mAbs tested. At a mAb to NS5B molar ratio of 4 the
polymerase function is inhibited almost completely. Starting from this point the cpm value is
approximating to the background radioactivity value of ~1500 cpm (not shown on the graph).
Control antibody 6G5.1 slightly varies the NS5B activity at molar ratios up to 1:1, however,
at the next points the activity remains more or less constant, indicating that there is no dose-
dependent inhibitory effect upon NS5B.
The F-2A 7G8 and 8B2 monoclonal antibodies, expressed from a single promoter,
were also analyzed in the RdRp inhibition assay. According to the experimental data,
recombinant 8B2_F2A mAbs behave similarly to the hybridomal 8B2 and, consequently,
similarly to above described recombinant 8B2H and 8B2M mAbs (Figure 23). On the
contrary, 7G8 F2A mAbs exhibit a very unexpected behaviour – both, chimeric and murine
antibodies show much greater potency to inhibit the NS5B polymerase in this assay, however,
still lesser than that of recombinant murine 7G8M antibody. Such an enhancement of
recombinant antibodies biologic activity might be possible due to several reasons. First, since
F2A antibodies are expressed from a single promoter resulting in 1:1 molar ratio of both
heavy and light chains a greater concentration of fully dimerized intact antibodies should be
produced. Whereas, in case mAbs are produced using two-gene cassettes, excess undimerized
heavy and/or light chains might interfere with the correct assembly of functional antibody.
Second, because F2A mAbs tended to precipitate during the buffer exchange these molecules
could also trigger the co-precipitation of NS5B and/or RNase inhibitor during the reaction,
and, consequently, diminish the synthesized radioactive RNA yields. The mAb precipitation
could also explain why the 7G8M_F2A antibody (whose sequence is identical to 7G8M mAb)
exhibits lower activity compared to that of 7G8M mAb. The tendency to precipitate, in its
turn, could be caused by incomplete furin cleavage of translated peptides and deficient folding
of the resulting proteins. In this case, the F-2A peptide sequence at the C-terminal part of the
heavy chain can probably bind to other antibody molecules and induce subsequent
precipitation, or physically interfere with antibody packing. On the other hand, in both cases
58
(precipitaion or low relative amount of functional antibody molecules) similar results should
have been probably observed also for 8B2 F2A mAbs, which, however, do not show any
significant improvement (as well as impairment) of their inhibitory activity. The presence of
two additional amino acids at the C-terminus of the antibody heavy chains does not seem to
be the reason also, because it is the terminal part of the constant domain, which is spatially
very far away from the antigen-binding site of the antibodies. Thus, for now the enhanced
inhibitory activity remains a puzzle for us, and additional experiments should be carried out,
in order to make sure, that such behaviour is not just an artefact, caused by the antibody
precipitation.
Thus, several conclusions can be made from these experiments:
1. The recombinant 7G8 and 8B2 mAbs work in the same manner as the
hybridoma mAbs, justifying the approach of antibody engineering applied in our study.
This approach might be exploited also for the construction of other chimeric antibodies of
interest.
2. 7G8_F2A antibodies exhibit an enhanced inhibitory activity, comparing to
hybridoma 7G8 mAb. The reason for this is unknown at present and additional
experiments are needed to reveal the nature of such unexpected behavior. At the same
time, 8B2_F2A antibodies show an inhibition pattern almost identical to that of
hybridoma 8B2 mAb, with only a slightly higher activity at lower mAb concentrations,
what might be a benefit of the one-promoter expression system.
3. The biologic activity of recombinant 7G8M antibody is higher than of its
chimeric and hybridoma analogues. This fact can be possibly explained by several
mutations in FR1 regions of heavy and light chain variable regions, introduced due to the
use of the degenerate primers. It is clear that the constant chains could not be the reason of
this enhancement, because mAbs 7G8H and 7G8.1, which have constant chains from
different sources, function identically to each other. Consequently, the variable regions
should be the case.
4. Another interesting point is the correlation of data obtained for 7G8 and 8B2
recombinant mAbs in ELISA and RdRp inhibition assay. RdRp assay is aimed in the first
place at testing the inhibitory activity of antibodies, whereas ELISA demonstrates the
affinity of the antibody to a given antigen. However, both affinity and biologic activity
have to be closely connected to each other. This is what we see, when comparing the
ELISA and RdRp assay results. 8B2M and 8B2H mAbs exhibit almost identical affinity to
59
NS5B and the same picture is seen in RdRp inhibition experiments – both mAbs are
equally efficient inhibitors in RdRp assay (Figure 23, 24). In case of 7G8H and 7G8M
mAbs it is evident that the inhibitory activity of 7G8M antibody is stronger than that of
7G8H mAb (Figure 24). At the same time, signal intensity obtained in ELISA assay for
7G8M antibody at the concentration of 0.1 ng/µl is higher than for 7G8H at the same
concentration (Figure 23), indicating the higher affinity of 7G8M to NS5B. These results
suppose that the higher affinity of 7G8M to NS5B makes it more efficient inhibitor,
compared to 7G8H (and to hybridoma 7G8, as can be judged from Figure 24).
Unfortunately, the hybridoma mAbs cannot be compared based on results of these
experiments, because different stocks of these antibodies were used in ELISA and RdRp
assays. At this point, it becomes clear that at least for 7G8M mAb higher affinity results in
higher inhibitory activity, and proves that results obtained for 7G8M in RdRp assay are
not an artefact.
3.1.8. Identification of mutations, possibly enhancing the biologic activity
of 7G8M mAb.
7G8H and 7G8M mAbs were constructed by us utilizing different strategies. Former,
was created by RF-cloning method (simultaneously modifying several amino acids in its FR1
regions, (see “methods”)). The latter, 7G8M, was prepared by utilizing degenerate primers,
which could, with high probability, modify several amino acids in the FR1 sequences of the
mAb variable regions (due to the PCR is biased method), specifically – 1, 3, 5 and 6-th amino
acids in VH and 4-th, 7-th and 8-th amino acids in VL (Figure 25).
Figure 25. Sequences of degenerate primers used in this study for amplification of antibody variable regions.
Posible amino acids that can be generate using these primers are shown in italics, above the respective nucleotide
triplets. Upper – VH primer, lower – VL primer. Restriction sites, introduced to the variable region sequences
during amplification are underlined.
60
The sequences of the variable regions of mAbs 7G8H and 7G8M were aligned and
carefully analyzed. The analysis revealed differences between 1st, 3
rd and 5
th amino acids of
VH regions, and between 3rd
and 4th
amino acids of VL regions of the antibodies (Figure 26).
Figure 26. Amino acid sequence alignment of chimeric and murine recombinant 7G8 antibody VH and VL
regions. Mismatching amino acids are shown in bold. Symbols in “consensus” line show the biochemical
similarity of mismatching amino acids. * means the same amino acids, : - high degree of similarity, and space –
completely dissimilar amino acids.
To the very best of our knowledge, there are no published works, showing that these
amino acids could be critical for the overall mAb conformation, or a conformation of its
CDRs. However, some of these residues are involved in the formation of β-sheet structures
through hydrogen bonding to other residues [8]. That is why we decided to graft these amino
acids from 7G8M antibody into the chimeric 7G8 mAb. If the assumption were correct, the
resulting antibody should get an increase in efficiency, similar to that of 7G8M antibody. This
would have allowed us, first, to construct a highly efficient chimeric antibody which could be
possibly used as a therapeutic agent in future, and second – to discover the amino acids,
which may play an important role in conformation of the antibody variable regions.
3.1.9. Exchanging FR1 regions of 7G8H antibody for those of 7G8M.
Grafting of the 7G8M FR1 regions to 7G8H chimeric construct was carried out using
the conventional restrictase-mediated cloning of the whole VH and VL regions (see
“Methods”). Although, this mAb was expressed from two cassettes, the expression gave an
impressive yield of 9 mg of antibody, what is ~2 times more, than it can be achievable using
hybridoma technology. This fact gives us a reason to suppose that mutations introduced to the
FR1 region of chimeric 7G8H antibody could somehow stabilize the variable domains of this
mAb, thereby increasing the stability of the whole immunoglobulin molecule and increasing
the amount of soluble antibody by lessening the risk of precipitation.
61
Summary
Two murine monoclonal antibodies inhibiting HCV RdRp in vitro were previously
identified and extensively described in our lab. The ability to inhibit the key enzyme in HCV
replication cycle demonstrates the therapeutic potential of these mAbs. As murine mAbs tend
to induce anti-immunoglobulin response in humans, they have to be appropriately modified
first and have to maintain their biologic activity after all modifications. Thus, two major tasks
were pursued in this study. First, development of chimeric HCV RdRp-specific mAbs with
preserved biologic activity. Second, development of a customizable chimerization strategy,
that could be used for rapid and reliable chimerization of virtually any other hybridoma-
produced mAbs.
Variable regions of mAbs 8B2 and 7G8 were successfully amplified from hybridoma
cDNA by utilizing degenerate primers. The sequences of the mAb variable regions were
determined by sequencing. Using the same degenerate primers strategy and unique restriction
endonuclease recognition site, a simple and reliable high-throughput system for amplification
of variable regions from hybridomas and joining them directly to human or mouse constant
regions was developed (together with Icosagen Group).
Using the above described system chimeric 7G8 and 8B2 genes, as well as
recombinant mouse 7G8 and 8B2 genes, were constructed and inserted into expression vector
(provided by Icosagen Group) utilizing two separate expression cassettes for both heavy and
light chains. Recombinant mAbs were produced utilizing novel QMCF technology developed
by Icosagen Group, yielding high amounts of protein – comparable and exceeding those
obtained using hybridoma technology.
Additional recombinant constructs of the same mAbs were generated, in which heavy
and light chain genes were connected with a short linker, encoding the furin cleavage site and
FMDV 2A peptide. These recombinant genes could be expressed from a single promoter,
producing mAb heavy and light chains at equimolar ratio, what resulted in approximately 2-
fold increase of antibody yields.
Sufficient amounts of wild type NS5B along with its inactive mutant form (GND)
were purified for use in biochemical experiments. Enzymatic activity of wtNS5B was
demonstrated in RdRp assay. Subsequently, all generated recombinant antibodies were tested
in RdRp assay for their ability to inhibit HCV NS5B polymerase. Antibodies produced using
62
two-promoter system were additionally tested in ELISA assay for their NS5B-binding
affinity. The results of these experiments clearly indicate that there is no loss of biologic
activity for any of the recombinant antibodies produced during this study compared to
hybridoma mAbs. Moreover, one of the recombinant murine mAbs, specifically 7G8M,
showed an enhanced inhibitory activity. This phenomenon was probably caused by several
mutations in its FR1 region, introduced by degenerate primers in PCR amplification stage.
These mutated amino acids were further grafted to the chimeric 7G8 antibody, in order to test
this hypothesis. For the moment, there is no data available on biologic activity of this
antibody and additional experiments still have to be done.
63
C hepatiidi viiruse RNA-sõltuvat RNA-polümeraasi inhibeerivate hiire
monokloonsete antikehade rekonstrueerimine ja bioloogiline aktiivsus
Aleksei Suslov
Resümee
Antikehad ehk immunoglobuliinid on heterodimeersed Y-tähe kujulised glükoproteiinid, mis
seovad spetsiifilisi märklaudmolekule – antigeene. Tüüpiline immunoglobuliini molekul
koosneb neljast polüpeptiidsest ahelast – kahest raskest ahelast (~50 kDa) ja kahest kergest
ahelast (~25 kDa). Raske ja kerge ahel, samuti nendest moodustunud dimeerid, on omavahel
ühendatud disulfiidsidemetega. Erineva spetsiifilisusega antikehade mõlema ahela N-
terminaalsed osad (esimesed ~110 aminohapet) varieeruvad oma järjestuses tugevasti, seega
on need regioonid tuntud kui V (variable) regioonid. Kergete ja raskete ahelate ülejäänud
piirkonnad on väga konserveerunud. Y-kujulise molekuli „käsi“ (ülemist avatud osa)
nimetatakse Fab fragmendiks, mis vastutab antigeeni sidumise eest. Molekuli „jalg“
omakorda vastutab rakus erinevate antigeenidega võitlemise mehhanismide aktiveerimise eest
ja seda nimetatakse Fc regiooniks (Fc retseptori sidumisala). V regioonid koosnevad kahest β-
lehest, mis asetsevad kohakuti. Need β-lehed koosnevad antiparalleelsetest β-ahelatest, mida
stabiliseerivad vesiniksidemed. β-ahelad on omavahel seotud erineva pikkusega lingudega,
mis moodustavad mõlema ahela V regioonide kolm kõige varieeruvamat piirkonda. Neid
linge nimetatakse hüpervarieeruvateks (hypervariable loop) ja tähistatakse raske ahela puhul
H1, H2, H3 ja kerge ahela puhul L1, L2, L3. Kristallstruktuuride analüüs näitas, et mõlema
ahela varieeruvad lingud moodustavadki antigeeni siduva saidi. Kuna need regioonid on
komplementaarsed vastava antigeeni struktuuriga, on need tuntud kui komplementaarsust
määravad regioonid (complementarity determining regions; CDR-id). Ülejäänud varieeruvate
regioonide osad moodustavad stabiilse karkassi, mis hoiab hüpervarieeruvaid regioone õiges
konformatsioonis. Teades aminohappejääke, mis vastutavad nende konformatsioonide
moodustamise eest, on võimalik kanda antigeenset spetsiifilisust ühelt antikehalt teisele ilma
aktseptor-antikeha funktsiooni rikkumata.
Antikeha V regioonide järjestusi kodeerivad geenid koosnevad erinevatest omavahel
juhuslikult rekombineeruvatest geenisegmentidest. Seepärast on võimalike kombinatsioonide
arv väga suur, tekitades suure hulga erineva spetsiifilisusega antikehasid. Varieeruvus ning
väga spetsiifiline ja tugev antigeeni sidumise võime muudavad antikehad terapeutilise
potentsiaaliga molekulideks. Selline idee oli juba ammu, kuid tehnoloogia hiire
monokloonsete (sama spetsiifilisusega) antikehade tootmiseks ilmus alles 35 aastat tagasi.
Tehnoloogia kasutusele võtmise järgselt selgus peagi, et hiire antikehad, manustatuna
inimesele, põhjustavad anti-immunoglobuliin immuunvastuse. Seega hakati arendama
erinevaid strateegiaid, mille eesmärgiks oli vähendada hiire antikehade immunogeensust,
muutes nende järjestusi rohkem inimeselaadseteks. Taoline lähenemine hõlmas kimäärsete
(hiire antikeha varieeruvad regioonid liidetakse inimese antikeha konstantsete regioonidega)
ja humaniseeritud (hiire antikeha CDR-id viiakse inimese antikeha struktuuri) antikehade
konstrueerimist. Viimane strateegia osutus edukaks ning tänapäeval on suurim osa turul
olevatest terapeutilistest antikehadest just humaniseeritud antikehad.
Ühed suure terapeutilise potentsiaaliga antikehad on rakusisesed antikehad. Kuna konstantsed
regioonid ei ole raku sees funktsioneerimiseks vajalikud, konstrueeritakse selliseid antikehi
põhiliselt scFv molekuli kujul. scFv koosneb kerge ja raske ahela varieeruvatest domäänidest,
mis on omavahel seotud peptiidse linkeriga. Viimasel ajal on rakusiseste antikehade
64
tuvastamiseks ja isoleerimiseks arendatud mitmeid tehnoloogiaid. Sellised antikehad (scFv
molekulid) sisaldavad konsensusjärjestust, mis tagab nende stabiilsuse, võimaldades
korrektset struktuuri isegi tavaliselt disulfiidsidemete moodustamist takistavas redutseerivas
rakusiseses keskkonnas. Viimase 20 aasta jooksul on konstrueeritud suhteliselt palju
rakusiseselt töötavaid antikehasid, kuid ükski neist ei ole veel jõudnud turule. Tehnoloogiad,
mis võimaldavad stabiilsete rakusiseste scFv-de isoleerimist, suudavad suure tõenäosusega
seda olukorda lähemas tulevikus muuta.
Antud magistritöö eesmärgiks oli kahe meie laboris eelnevalt kirjeldatud C hepatiidi
viiruse RNA-sõltuvat RNA-polümeraasi inhibeeriva hiire 7G8 ja 8B2 antikeha varieeruvate
regioonide kloneerimine hübridoomi cDNA-st, nende antikehade rekombinantsete kimäärsete
variantide konstrueerimine ja bioloogilise aktiivsuse määramine.
Tuginedes eksperimentaalsel teel saadud andmetele, võib töö tulemused kokku võtta
järgnevalt:
7G8 ja 8B2 varieeruvad regioonid kloneeriti hübridoomi cDNA-st ja liideti kokku kas
inimese või hiire konstantsete regioonidega, genereerides nii kimäärseid kui natiivseid
rekombinantsete antikehade geene. Selleks loodi lihtne ja efektiivne vektor-praimer süsteem,
mis võimaldab amplifitseerida varieeruvaid regioone ükskõik millisest hübridoomist ja liita
need inimese või hiire konstantsete regioonidega.
Konstrueeritud rekombinantsed geenid kloneeriti ekspressioonivektoritesse kahel
viisil: esimesel juhul ekspresseeriti antikeha ahelad kahe eraldi promootori alt, teisel juhul
ühendati mõlema ahela geenid omavahel lühikese järjestusega, mis kodeeris furiini lõikesaiti
ja Foot-and-mouse disease viiruse 2A peptiidi. Ahelate ühendamine võimaldas ekspresseerida
mõlemat geeni ühe transkriptsiooniühikuna, seejuures vahelejääv linkerala tegi võimalikuks
nende post-translatsioonilise eraldamise. Sellise ühe promootori alt ekspresseeritud kerge ja
raske ahela molaarne suhe on 1:1.
Antikehad toodeti, kasutades QMCF tehnoloogiat (välja töötatud Icosagen Group’i
poolt). Ühe promootori alt ekspresseerides oli antikeha saagis umbes 2 korda suurem kui kahe
promootori alt ekspresseerides ja ligikaudu 1,5 korda suurem kui hübridoomi tehnoloogia
puhul.
Metsiktüüpi NS5B RNA polümeraas ja selle inaktiivne mutantne vorm (GND)
puhastati piisavas hulgas ja metsiktüüpi NS5B ensümaatiline aktiivsus näidati in vitro RNA
sünteesi reaktsioonides.
Konstrueeritud antikehade võime blokeerida HCV polümeraasi aktiivsust kontrolliti in
vitro RNA sünteesi katsetes. Kahe promootori kasutamisel ekspresseeritud antikehade
afiinsus NS5B polümeraasile kontrolliti lisaks ka ELISA testi abil. Saadud tulemused
näitavad selgelt, et rekombinantsete antikehade aktiivsus ja afiinsus ei ole langenud ja veelgi
enam, rekombinantse hiire 7G8 antikeha puhul täheldati isegi inhibiitoorse aktiivsuse tõusu,
mis võiks olla seletatav selle antikeha FR1 regioonidesse degenereeritud praimeritega PCR-i
käigus sisestatud mutatsioonidega. Tõestamaks (või ümber lükkamaks) seda hüpoteesi (et just
need mutatsioonid on muutnud antikeha aktiivsust), olid muteerunud aminohapped ümber
tõstetud kimäärse 7G8 antikeha sisse. Praeguseks hetkeks eksperimentaalsed andmed selle
antikeha funktsionaalsuse kohta veel puuduvad.
65
Acknowledgements
This work was carried out at Professor Mart Ustav’s lab at the chair of Microbiology
and Virology, Institute of Technology, Estonia, in collaboration with Icosagen Group. There
is a lot of people who contributed to a greater or lesser extent to this work, which definitely
would not be done without all of them. I would like to thank:
First of all, my supervisor Andrei Nikonov, for introducing me to the world of
Science, teaching me a lot of things, especially to work independently being at the same time
a part of a group and to be accountable for my actions and decisions. Also, for stimulating
talks and keeping me motivated on my way to a master’s degree. You have changed my view
of many things.
My professor Mart Ustav for giving me possibility to work in Your team and for
interesting projects and ideas.
Icosagen team – Andres Tover, Radi Tegova, Anne Kalling, Andres Männik, Polina
Verhovtsova – for technical help, interesting solutions and of course for production of all of
these antibodies in large quantities.
Erkki Juronen – immunogeneetika ja -keemia vanemteadur at General and Molecular
Pathology Institute, – for your co-operation, helpful advices and perfectly working mAbs.
My colleagues – Mihkel Allik – for teaching me various tips and tricks, and for
valuable discussions and advices; also Nele Jaanson, Axel Soosaar, Vallo Varik.
Colleagues from other labs – Joachim Gerholdt – for providing me with always fresh
isotopes; Sergei Kopantšuk – for making my radioactive life easier; also Mardo Kõiv, Ervin
Valk, Evgeni Mihheev for various assistance.
Special thanks to Ly Käsper, Age Utt, Sirle Saul, Teele Karafin for enriching our floor
with these positive vibrations.
Karin Lauga – for reviewing the estonian part of this work.
My friends – for your support and inspiration.
And, of course, my parents for beeing help in all times.
66
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