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COMPLEMENT-MEDIATED KILLING OF CANCER CELLS
Cover: fluorescence microscopical image of cryostat sections of ovarian
microtumors treated with antibodies and complement. Cells with yellow
fluorescence are killed by complement.
© 2003 by Juha Hakulinen
Printed at Yliopistopaino, Helsinki, Finland
ISBN 952-91-5503-4 (sid.)
ISBN 952-10-0884-9 (pdf)
http://ethesis.helsinki.fi
COMPLEMENT-MEDIATED KILLING OF CANCER CELLS
JUHA HAKULINEN
Haartman Institute
Department of Bacteriology and Immunology
University of Helsinki
Finland
Academic Dissertation
To be publicly discussed, with the permission of the Medical Faculty of the
University of Helsinki, in the small Auditorium of the Haartman Institute,
Haartmaninkatu 3, Helsinki, on Saturday, January 11th, 2003, at 12 o´clock
noon.
SUPERVISOR
Seppo Meri
MD, PhD, Professor
Haartman Institute
Department of Bacteriology and Immunology
University of Helsinki
REVIEWERS
Mikko Hurme
MD, PhD, Professor
University of Tampere Medical School
and
Timo Paavonen
MD, PhD, Docent
Department of Pathology
University of Helsinki
OPPONENT
Olli Lassila
MD, PhD, Professor
Department of Medical Microbiology
University of Turku
CONTENTS
ORIGINAL PUBLICATIONS...............................................................................................7
ABBREVIATIONS..............................................................................................................8
ABSTRACT .................................................................................................................... 1 0
1. INTRODUCTION........................................................................................................ 1 2
2. REVIEW OF THE LITERATURE................................................................................. 1 4
2.1 The complement system................................................................................. 1 4
2.1.1 Overview ..................................................................................................... 1 4
2.1.2 The alternative pathway.......................................................................... 1 6
2.1.3 The classical pathway.............................................................................. 1 7
2.1.4 The lectin pathway.................................................................................... 1 8
2.1.5 The lytic pathway...................................................................................... 1 8
2.1.6 Soluble regulators of complement......................................................... 2 0
2.1.7 Membrane-bound complement regulatory proteins (mCRP)............. 2 1
2.1.8 Rat complement system.......................................................................... 2 6
2.2 The immune system and transformed cells................................................. 2 7
2.2.1 Antigen-presenting cells (APC)................................................................ 2 7
2.2.2 T lymphocytes ........................................................................................... 2 8
2.2.3 Natural killer cells (NK cells).................................................................... 2 9
2.2.4 Tumor antigens ......................................................................................... 3 0
2.2.5 Antibody responses against tumor-associated antigens (TAA) in
cancer patients in vivo.......................................................................... 3 0
2.2.6 Antibody-induced effector mechanisms................................................ 3 1
2.2.7 Tumor antigens and immunotherapy of cancer.................................. 3 3
3. AIMS OF THE STUDY .............................................................................................. 3 7
4. MATERIALS AND METHODS .................................................................................. 3 8
5. RESULTS AND DISCUSSION................................................................................... 4 1
5.1 mCRP on tumor cells and milk fat globules (MFG; I, II, III, IV)................... 4 2
5.1.2 Characterization of CD59 on T47D cells and MFG (I, II) ................... 4 4
5
5.2 Complement-mediated killing of MCF7 and T47D cells (I)......................... 4 6
5.2.1 Expression of TAA on ovarian cancer cells (III) .................................. 4 8
5.2.2 Complement-dependent cytotoxicity (CDC) and ovarian cancer cells
(III)............................................................................................................ 4 9
5.3 CDC and microtumor spheroids (MTS; IV)................................................. 5 0
5.3.1 The penetration of mAb and complement into MTS .......................... 5 4
5.4 A rat model to study complement activation in vivo (V)......................... 5 6
5.4.1 CDC and rat colorectal cancer cells...................................................... 5 7
5.4.2 C3 deposition on CC531 cells and CDC................................................. 5 7
5.4.3 Complement activation in situ................................................................. 5 8
5.4.4 Homing of mAb into tumors and complement activation in vivo..... 5 9
6. CONCLUSIONS.......................................................................................................... 6 1
7. ACKNOWLEDGMENTS.............................................................................................. 6 3
8. REFERENCES............................................................................................................. 6 5
6
ORIGINAL PUBLICATIONS
This thesis is based on the following original articles, which are referred to in
the text by their Roman numerals. Some unpublished data are also presented.
I
Hakulinen J and Meri S. (1994).
Expression and function of the
complement membrane attack complex inhibitor protectin (CD59) on
human breast cancer cells. Lab Invest 71, 820-827.
II
Hakulinen J and Meri S. (1995).
Shedding and enrichment of the
glycolipid anchored complement lysis inhibitor protectin (CD59) into milk
fat globules. Immunology 85, 495-501.
III
Bjørge L, Hakulinen J, Wahlström T, Matre R and Meri S. (1997).
Complement regulatory proteins in ovarian malignancies. Int J Cancer
70, 14-25.
IV
Hakulinen J and
Meri S. (1998).
Complement-mediated
lysis
of
microtumors in vitro. Am J Pathol 153, 845-855.
V
Gelderman K, Hakulinen J, Hagenaars M, Kuppen P, Meri S and Gorter A..
(2002).
Membrane-bound
complement
regulatory
proteins
inhibit
complement activation by immunotherapeutic mAb in a syngeneic r a t
colorectal cancer model. Submitted.
The articles in this thesis have been reproduced with the permission of the
copyright holders: the United States and Canadian Academy of Pathology (I),
Blackwell Publishing (II), John Wiley & Sons Inc. (III) and the American Society
of Investigative Pathology (IV).
7
ABBREVIATIONS
51 Cr
Chromium-51
125 I
Iodine-125
ADCC
antibody-dependent cellular cytotoxicity
AF
ascitic fluid
Ag
antigen
BSA
bovine serum albumin
C4bp
C4b binding protein
C9DS
human serum deficient in C9
CD
cluster of differentiation
CDC
complement-dependent cytotoxicity
CDCC
complement-dependent cellular cytotoxicity
CD35
complement receptor type 1 (CR1)
CD46
membrane cofactor protein (MCP)
CD55
decay-accelerating factor (DAF)
CD59E
CD59 isolated from human erythrocytes
CD59M
CD59 isolated from breast milk
CD59U
CD59 isolated from urine
CD95
Fas receptor
CR1
complement receptor type 1 (CD35)
DAF
decay-accelerating factor (CD55)
ECL
electrochemiluminescence
FITC
fluorescein isothiocyanate
GPE
guinea pig erythrocyte
GPI
glycosyl-phosphatidylinositol
IF
immunofluorescence
kDa
kilodalton
mAb
monoclonal antibody
MAC
membrane attack complex of complement
MASP
mannose binding lectin associated serine protease
MBL
mannose binding lectin
MCP
membrane cofactor protein (CD46)
8
mCRP
membrane-bound complement regulatory protein
MFG
milk fat globule
Mr
relative molecular weight
MTS
microtumor spheroid
NHS
normal human serum
NK-cell
natural killer cell
NP40
Nonidet P40
OD
optical density
pAb
polyclonal antibody
PBS
phosphate buffered saline
PI
propidium iodide
PIPLC
phosphatidylinositol-specific phospholipase C
PIPLD
phosphatidylinositol-specific phospholipase D
SC5b-8
soluble C5b-8 complex
SDS-PAGE
sodium dodecyl sulphate polyacrylamide gel
electrophoresis
TAA
tumor associated antigen
TCC
terminal complement complex
VBS
veronal buffered saline
9
ABSTRACT
The human blood complement system is a part of the innate immune system.
The principal function of complement is to defend the host against microbial
infections and other foreign material. Furthermore, complement participates in
the clearence of apoptotic cells and immune complexes from the blood and
other tissues. The activation of complement by antibodies or by a foreign
surface results in opsonization, chemotaxis and direct killing of microbes and
infected cells. In the host, complement activation causes inflammation and
tissue damage. Host cells express complement regulators (CD46, CD55 and
CD59) on their surfaces to restrict harmful complement activation on the cells
to a minimum. However, the same molecules also make complement-mediated
destruction of malignant cells difficult. The purpose of the current study was
to respond to this challenge by using antibodies and complement as an
effector
mechanism to
destroy
malignant
cells and
by
analyzing
the
significance of the membrane bound complement regulator CD59 for the
cancer cell survival from
complement
complement resistance of breast
attack.
In the
first
study,
the
cancer cells (MCF-7 and T47D) was
examined. It was possible to increase complement-mediated lysis of these cells
by inactivating CD59 on the cell membranes with a specific monoclonal
antibody (I). During the first study it was noticed that CD59 was strongly
stained in the lumina of milk ductules in sections of breast cancer tissue. This,
and the fact that CD59 had been previously detected in the human breast
milk, led to the observation (II) that CD59 in milk is associated with structures
called milk fat globules (MFG). In the third study (III) complement regulators
CD46 and CD59 were found to be strongly expressed on ovarian cancer cells.
Furthermore, it was possible to kill ovarian cancer cells using NHS as a source
of complement after sensitizing the cells with a mAb reacting with an ovarian
tumor associated antigen and neutralizing CD59 on cell surface with specific
mAb.
Although it was possible to use complement and antibodies to destroy
individual breast cancer cells in suspension, many cancers exist as multicellular
10
tumors that are more resistant to complement-mediated killing than single
cells1. To analyze factors that are associated with killing of cells growing in
three-dimensional
aggregates
multicellular
microtumor
spheroids
(MTS)
established from breast carcinoma (T47D) and ovarian teratocarcinoma (PA1) cell lines were used as models to study complement-mediated destruction
of small solid tumors (IV). This study showed differences in penetration o f
complement components and monoclonal antibodies into the tumor tissue.
Furthermore, complement attack against MTS and the neutralization of CD59
on the cell membranes resulted in a reduction of the microtumor volume. The
microtumor spheroid study suggested that complement-mediated killing o f
cancer cells would be effective against individual cells or small clusters o f
malignant cells that may remain after surgical removal of the main tumor. In
the last study
(V),
rat
colorectal
cancer cells (CC531) were injected
subcapsularly into the liver of Wag/Rij rats as a model for metastases o f
colorectal carcinoma. A panel of specific mAbs to CC531 cells were tested f o r
their complement-activating and tumor-homing capacities in vivo. In this study
it was possible to kill the CC531 cells in vitro using mAb and rat serum and t o
activate complement on tumor sections in situ. However, no C3 deposition
was detected on the tumors
in vivo indicating a role for
complement
regulators.
In conclusion, these studies show that it is possible to destroy single malignant
cells or partially even microtumors with serum complement if the complement
regulator
CD59 is inactivated with a specific mAb on the cancer cell
membranes. In addition to cell membranes the glycophospho inositol-anchored
form of CD59 was detected on MFG in vivo. Complement-mediated killing o f
MTS showed that antibodies and C1q are able to penetrate through the
microtumor spheroids but the penetration of C3 is restricted by its strong
activation and covalent binding to cell surfaces. The syngeneic rat model
suggested that complement-activating mAb penetrate into the tumor tissue
but the lack of penetration of C3 into the tumor is a problem also in vivo.
11
1. INTRODUCTION
A quarter of the western world population faces the fact that they get a
malignant tumor
during their life time. Although cancer therapies have
improved a lot during the last decades cancer is still fatal for most people.
Immunotherapy has only been little used for the treatment
of cancer.
However, it offers one potential approach. Immunotherapy for cancer was
first used as early as 1895 by Hericourt and Richet who attempted to t r e a t
cancer patients with antitumor antisera prepared in dogs and donkeys.
However this and many other attempts
failed to cure the patients. The
invention of the hybridoma technique by Köhler and Milstein in 1975 2 and the
discovery of new cancer antigens have given new hope for the specific
destruction of tumor cells by monoclonal antibodies (mAb). Much of the
research to improve the cytotoxicity of mAb has focused on conjugating them
with toxins or radionuclides. However, an ideal immunotherapeutic tumor-killing
system would harness the patient´s
own effector
mechanisms such as
antibody-dependent cellular cytotoxicity (ADCC) or the complement system t o
destroy the malignant cells. Complement belongs to the innate immune system
that, as opposed to the acquired immune system constitutes the nonadaptive
part of the human immune system. The immune system recognizes and
removes foreign material (viruses, bacteria and other micro-organisms) and is
responsible for the clearance of tissue debris resulting from ageing cells o r
trauma. Discrimination of foreign structures from the normal host tissue
components is the key element in both systems. This leads to activation o f
specific mechanisms to
eliminate microbes or
non-viable cells. The key
components of adaptive immune system are lymphoid cells (B and T cells).
They recognize their targets by multiple specific B cell and T cell receptors.
The acquired immune response is slow (starting from 3 - 5 days) because o f
the need for clones of responding B and T cells to develop. However, the
acquired immunity develops a memory from previous infections. The innate
immunity includes serum complement, natural killer cells and phagocytic cells.
The innate immunity recognizes molecular structures in microbes by receptors
with
broad
specificity.
The
structures
12
can
be
polysaccharides
and
polynucleotides that are common in microbes but often not found in the host.
An example is bacterial
lipopolysacharide (LPS) that
directly activates
complement and, when in complex with LPS-binding protein, is recognized by
the macrophage receptor CD14. Because of the broad specificity of the
receptors and no need for clonal expansion the response of the innate
immunity is immediate. Innate and acquired immunities are not completely
separate systems. Innate immunity can trigger adaptive immunity and vice
versa. Phagocytes (macrophages
and dendritic cells) present
microbial
antigens to T cells to initiate both cell-mediated and antibody-mediated
adaptive immune responses. Similarly, the antibodies produced by adaptive
immunity can activate the classical pathway of complement against a specific
antigenic structure.
While microbes are often
promptly
destroyed
by the immune system,
developing tumor cells are usually not. Cancers result from the outgrowth of a
single malignant host cell. Immune system has to discriminate the malignant
cells from the normal host cells in order to destroy them. However, except f o r
virus-induced tumors there are often no or only few antigenic differences
between normal and malignant cells. The rejection of cancer cells by immune
system is based on tumor-associated antigens (TAA) that are not expressed
on normal cells or their expression is lower. The success of using complement
against malignant cells requires a specific activation of complement against
these
cells with
mAbs
or
other
molecules that
recognize
TAA
and
understanding of the mechanisms of complement regulation on cancer cells.
13
2. REVIEW OF THE LITERATURE
2.1 The complement system
2.1.1 Overview
Complement is an essential part of the innate immune system. It co-operates
with the adaptive immune system by mediating inflammatory consequences o f
antigen-antibody interactions and contributing to the enhancement of the
humoral response mounted against specific antigens3, 4 . The principal function
of complement is to defend the host against microbial invasion of the body.
Furthermore, complement has an important role in the disposal of dead and
apoptotic
cells and immune complexes 5-7. Complement can discriminate
between self and non-self structures but not as specifically as the acquired
immunity.
The complement system consists of a complex group of proteins that are
present in blood plasma and on cell membranes. The proteins act as
precursor
enzymes, effector
molecules (Table
1),
control
proteins
or
receptors (see chapters 2.1.6 and 2.1.7). Most of the complement proteins
circulate in the bloodstream and body fluids as inert precursors. The contact
of the first component with an activator i.e. an immunoglobulin, activating
surface or certain carbohydrate structures, leads to a subsequent activation
of the second one in a precise order depending on the pathway – classical,
alternative or lectin - that is activated. During the activation some of the
proteins acquire enzymatic properties while the others function as effector
molecules and control proteins. C3 is a key-component of the complement
system, since it can be activated through all three pathways3 .
The activation of complement results in three main types of biological
responses. The C3b and iC3b fragments 8 generated on the target surface
can interact with cell surface receptors of phagocytes to induce complement-
14
dependent cellular cytotoxicity (CDCC)9, 10 . The small cleavage fragments
C3a, C4a and C5a are released into the fluid phase. These small bioactive
peptides act as chemotaxins, leukocyte activators and as anaphylatoxins 11,
12 .
Finally, activation of the terminal complement pathway results in the
formation of the membrane attack complex of complement (MAC) on the
target cell membrane 13. Because complement is a cytolytic system and since
the pathways include enzymatic events that allow considerable amplification
during activation, the complement cascade must be carefully regulated t o
prevent damage to the host cells. The stringent control mechanisms include
multiple regulatory proteins in blood plasma and on cell membranes. Because
of this, normal host cells can usually resist the cytolytic activity of homologous
or autologous complement14 .
15
Table 1. Proteins involved in complement activation
Protein
Molecular
weight
(kDa)
Classical pathway
C1q
410
Serum
conc.
(µg/ml)
Function
70
C1r
C1s
C2
C4
34
31
25
600
Binds IgG and IgM; initiates the
classical pathway.
Cleaves / activates C1s
Cleaves / activates C4 and C2
Cleaves / activates C3 and C5
Binds C2 during activation
85
85
95
206
Lectin pathway
MBL
96 x 2-6
5
MASP-1
MASP-2
-
Binds to carbohydrates:
initiates lectin pathway
Activates MASP-2
Cleaves C2 and C4
225
1
25
Cleaves / activates C3 and C5
Cleaves / activates factor B
Stabilizes C3bBb convertase
Alternative
Factor B
Factor D
Properdin
83
pathway
100
25
153
Common for the pathways above
C3
195
1200
Lytic pathway
C5
180
85
C6
128
60
C7
C8
C9
120
150
79
60
55
60
Subunit in alternative pathway
C3/C5 convertase. Binds C5 in
convertases. Opsonisation
C5b: initiates membrane attack;
C5a is the major chemotactic/
anaphylatoxic peptide
C6, C7 and C8 associate with
C5b to form a membrane site
to which C9 can bind
Multiple C9 proteins polymerize
to generate transmembrane
pore
2.1.2 The alternative pathway
The alternative pathway is a phylogenetically older system than the classical
pathway 3 . The alternative pathway of complement is triggered by activation
16
of the C3 protein. C3 has an internal thiolester bond that undergoes cleavage
at a slow rate during a hydrolysis reaction with water. The product of this
reaction is C3(H2 O), which can form an initial C3 convertase by binding factor
B which becomes cleaved to Bb by factor D15 . The C3(H2O)Bb convertase will
cleave C3 to C3a and C3b. The cleavage product C3b can bind to the
microbial or host cell surface by forming a covalent linkage with -OH or -NH2
groups through the exposed thiolgroup. Recently, it has been shown t h a t
phosphatidyl serine on apoptotic cells can activate the alternative pathway 7 .
C3b binds the complement component factor B to form C3bB, Mg 2+ is
required as a catalyst for the reaction. The newly formed complex is a
substrate for the plasma enzyme factor D that cleaves C3bB to generate
C3bBb, the principal C3 convertase, which can cleave C3 to produce more C3b
and C3a. The unhampered operation of the C3 convertase leads to the
deposition of large numbers of C3b molecules on the microbial surface 16 .
Finally, a C5 convertase is generated when an additional C3b molecule is
recruited to the C3bBb complex. Cleavage of C5 by the C5 convertase
releases C5a and C5b and initiates the lytic pathway.
2.1.3 The classical pathway
The classical pathway of complement is initiated by the interaction of the C1q
subcomponent
of C1 with at
least two
Fc regions
of
antigen-bound
immunoglobulins (IgG or IgM). Additional activators the classical pathway are
the C-reactive protein 17 , the serum amyloid P component 18 and membrane
blebs on apoptotic cells19. Conformational change in C1q upon binding leads
to the activation of the C1r protease. C1r will proteolytically cleave C1s into
an enzymatically active form. C1s splits C4 exposing a nascent thiolester bond
in the cleavage product C4b. This enables C4b to attach covalently to the cell
surface. C4b has a binding site for C2 and C1s acts on the formed C4b2
complex to create C4b2a, the classical pathway C3 convertase. From this on
the cascade continues similarly to the alternative pathway with one molecule
17
of C3b bound to C4b2a to generate the C5-cleaving enzyme for the initiation
of the lytic pathway.
2.1.4 The lectin pathway
It has been shown that the classical pathway can also be triggered without
antibodies by mannose binding lectin (MBL), a C1q-like molecule that binds t o
mannose and N-acetylglucosamine structures
that
are present
on the
surfaces of microbes 20 . MBL forms a C1-like complex with serine proteases
MASP-1 and MASP-2. Conformational change in MBL upon binding leads to the
activation of MASPs which cleave C2 and C4 21, 22 . The cascade continues like
the classical pathway.
2.1.5 The lytic pathway
The formation of the C5-convertase through the either alternative or the
classical pathway initiates the membrane attack that results in the generation
of a large aggregate of proteins, the so-called membrane attack complex
(MAC) on the activating surface13 . The lytic pathway consists of five proteins
(C5b, C6, C7, C8 and C9) that are present in plasma. Sequential addition o f
C6, C7 and C8 to C5b generates C5b-8, which catalyzes the polymerization o f
C9 to a pore-like structure on the target membrane. The diameter of the
membrane channel varies between 10 Å and 150 Å 23 . Deposition of C5b-9
complexes on the membranes of nucleated cells results e.g. in the leakage o f
adenine nucleotides ATP, ADP and AMP. Furthermore, the exposure results in
an increase of intracellular Ca 2+ and the loss of mitochondrial membrane
potential 24 . Normally, killing of nucleated cells requires several C5b-9 lesions
per cell25, although C5b-8-mediated cell lysis has also been reported26.
18
CLASSICAL PATHWAY
ALTERNATIVE PATHWAY
LECTIN PATHWAY
IgG, IgM
Act ivat ing surfaces
Carbohydrat es
C1q
C3( H2 O) Bb
MBL
C1r
C1s
C4
C2
C4b2a
MASP1
MASP2
C4
C2
C4b2a
C3
C3a
C4a
C4a
C3b
B
D
P
C3bBb
C5
C5a
C5b678 (9) n MAC
Figure
1 . A schematic model illustrating the activation
complement. The classical pathway
is activated
pathways
of
by immune complexes
containing IgG or IgM and the alternative pathway is triggered directly by the
microbial surface. The lectin pathway is activated by a surface containing
mannose or N-acetyl glucosamine residues. The pathways
lead to
the
generation of the membrane attack complex (C5b-9 or MAC) on the cell
surface which damages the cell membrane and kills the microbe. Dashed lines
indicate enzymatic activity. For abbreviations see Table 1.
19
2.1.6 Soluble regulators of complement
To
prevent
excessive
or
inappropriate
consumption
of
complement
components the activation needs to be carefully controlled. At least six
regulatory proteins are present soluble in blood plasma (Table 2). The plasma
protein C1 inhibitor (C1 INH) binds to the activated
C1r2C1s2 enzyme
complex and prevents the cleavage of C4 and C2 by blocking the serine
esterase activities of C1r and especially of C1s 27. The C3 convertase C4b2a
is regulated by the serum C4b-binding protein (C4bp), which competes with
C2a for binding to C4b and displaces C2a from the complex28 . C4b in complex
with C4bp is susceptible to cleavage by the plasma protein factor I, another
serine esterase enzyme of complement. Recently, it has been suggested t h a t
ovarian cancer cells bind C4bp, which protects the cells from complement
lysis29. Mechanisms analogous
to
those
described
above
control
the
alternative pathway C3 convertase. The plasma protein factor H competes
with factor B and readily displaces Bb from the C3bBb convertase. Factor H
also forms a complex with C3b catalyzing its proteolytic destruction by
factor I30 . Two plasma proteins, clusterin (SP40,40) 31 and vitronectin (Sprotein) 32 bind to the forming C5b-7, C5b-8 and C5b-9 complexes to prevent
their insertion into cell membranes.
20
Table 2. Soluble regulators of complement
Protein
C1 inhibitor
Molecular weight
(kDa)
105
Function
Factor H
150
Inhibition of C3bBb
formation. Decay
dissociation of C3bBb.
Cofactor in C3b cleavage
C4b binding protein
540
Cofactor in C4b cleavage
Factor I
88
Cleavage of C3b, iC3b and
C4b
Vitronectin
(S-protein)
80
Keeps TCC* in solution
Inhibitor of C1r and C1s
Keeps TCC* in solution
Clusterin
70
(SP40,40
Apo-J)
*TCC, terminal complement complex
2.1.7 Membrane-bound complement regulatory proteins (mCRP)
Activated complement may also be toxic to the host cells. However, normal
human cells can resist the cytolytic activity of complement by expressing
several regulatory proteins. Four proteins are expressed on cell membranes
(Table 3). The membrane-bound regulators are species-selective protecting
the cells from homologous or autologous complement 33, 34 . The proteins are
clustered into groups by their reactivity with monoclonal antibodies (CD:
cluster of differentiation).
21
Table 3. Membrane-bound regulators of complement
Protein
Molecular
Function
Weight (kDa)
CD35, CR1
190, 220
Control of C3bBb/C4b2a
Cofactor in C3b and C4b inactivation
CD46, MCP
58-68
Cofactor in C3b and C4b
inactivation
CD55, DAF
70
Decay of C3/C5 convertases
CD59, protectin
18-25
Inhibition of MAC
The complement receptor type 1 (CR1, C3b receptor, C D 3 5 ) 35 is a
transmembrane protein. It dissociates the key enzymes of the complement
cascade, the C3 and C5 convertases, and promotes proteolytic inactivation o f
C3b and C4b by the plasma serine protease factor I. Factor I cleaves the
alpha chains of C4b and C3b to generate iC4b and iC3b which are incapable o f
forming classical (C4b2a) or alternative (C3bBb) pathway C3 convertases.
Unlike the other cofactors, CR1 acts as a cofactor also in the cleavage o f
iC3b to C3c and C3dg. CR1 is expressed on erythrocytes,
neutrophils,
monocytes, B lymphocytes, eosinophils and some T cells36. On the surface o f
phagocytes CR1 functions also as a receptor for C3b, iC3b, C4b and C1q thus
having a role in the clearance of complement-activating immune complexes37.
Membrane cofactor protein (MCP, C D 4 6 ) . MCP binds to accidentally o r
spontaneously deposited C3b and C4b molecules on cell surfaces and
promotes their inactivation by serving as a cofactor for factor I38 .
MCP is
present on almost all cells except erythrocytes.
Decay-accelerating
attached
to
factor
(DAF,
cell membranes with a
C D 5 5 ) is a glycoprotein
glycosyl-phosphatidyl
that
is
inositol-(GPI-)
anchor. DAF dissociates the C3 convertases releasing C2a and Bb from the
complexes39. DAF is expressed on most cells including erythrocytes37.
22
Protectin (CD59) was first isolated from human erythrocyte membranes 40,
41 .
CD59 (Fig. 2) has been also referred to as MACIF42, HRF-2043 and MIRL
44 .
To simplify the nomenclature the functional name protectin for CD59 has
been proposed 45 . The first direct experimental demonstration of a MAC
inhibitor on human erythrocytes was published in 1988 by Sugita et al.41 . The
primary function of CD59, inhibition of the membrane attack
complex o f
complement, was shortly established in many laboratories 40, 43, 44 . CD59
blocks formation of MAC by preventing the C5b-8 catalyzed insertion of C9
into lipid bilayers (Fig. 3) 45, 46 . In addition to erythrocytes, CD59 is widely
distributed on all other human blood cells40 as well as on endothelial and
epithelial cells of several organs 47 . CD59 has also been detected on cultured
endothelial cells48, 49 glomerular epithelial cells50 and spermatozoa 51 . CD59 is
anchored to cell membranes via its GPI-moiety. Soluble, hydrophilic forms o f
CD59, that lack the anchor phospholipid52 have been detected in various body
fluids such as urine, tears and saliva 40, 53 and have also been produced in
recombinant form54 . In addition to various cell membranes phospholipid-tailed
CD59 has been found in amniotic fluid55 and in seminal plasma, where it has
been
shown
to
be
associated
with
prostasomes 56 .
23
extracellular
organelles
called
21
C
N
C
C
1
10
C
C
32
C
43
C
66
55
C
C
C
77 N
PIPLC
PIPLD
(R)
RR
Amino acid
Ethanolamine
Phosphorus
N-acetyl galactosamine
N-acetyl glucosamine
Fucose
Mannose
N-glucose
Inositol
Galactose
N-acetyl neuraminic acid
PIPLC Phosphatidylinositol specific phospholipase C
PIPLD Phosphatidylinositol specific phospholipase D
R Acyl chain
Figure 2 . A schematic model of the polypeptide backbone, oligosaccharide
side chain and GPI-anchor side chains of CD59. The GPI-anchor contains two o r
three acyl chains that anchor the protein to the cell surface.
24
C9
A
C6 C7
C5b
Cell surface
C8
MAC
B
CD59
Figure 3 . Formation of the membrane attack
complex (MAC; C5b-9) o f
complement on the cell membrane (A) and its inhibition by CD59 (B). On the
membranes of foreign targets C5b-8 catalyzes the polymerization of multiple
C9 molecules to form a pore-like lesion (MAC) into the cell membrane. On the
host cells CD59 inhibits the insertion of C9 by binding to C8 (B).
25
2.1.8 Rat complement system
The complement activation pathways among vertebrates are relatively well
conserved. The complement regulatory system in rat
resembles that
of
humans: the rat analogues of human CD46, CD55 and CD59 have been
characterized 57-59 . In addition, a distinct C3 regulator, Crry/p65, has been
identified in rodents. Crry/p65 has both decay-accelerating and cofactor
activity for the C3/C5 convertases. Thus, Crry/p65 restricts activation o f
both the alternative and the classical complement pathway 60-62 . The mCRPs
are species-selective and protect
cells primarily only against homologous
complement. The species selectivity of mCRP requires a syngeneic animal
model to study the role of mCRP in vivo. Xenografts usually induce a massive
activation of complement in the recipient blood against the vulnerable graft 63 .
The hyperacute rejection is caused by complement-activating xenoreactive
antibodies to endothelial cells64. Human xenoreactive antibodies against pig
xenografts, for example, consist mainly of anti-Galα1-3Gal antibodies, which
occur in IgM, IgG and IgA classes64.
26
2.2 The immune system and transformed cells
The human body continuously confronts
foreign material
like potentially
pathogenic bacteria and viruses from the enviroment. However, cell-mediated
and humoral immune responses can in most cases discriminate the pathogens
from the host cells and destroy them. Cancers result from the outgrowth o f
genetically transformed cells. It has been difficult to show that the immune
system responds strongly to tumor cells. Mice that lack lymphocytes o r
humans that are deficient in T cells do not differ much in tumor incidence
compared to normal hosts 65 . Virus-associated tumors are an exception and
can be usually destroyed by the normal immune system 65 . However, in animal
studies some tumors have elicited immune responses that
prevent their
growth. In these studies the tumors have been induced by carcinogenic
chemicals or radiation in inbred strains of animals. The experimental tumors
can be isolated, grown in vitro and injected into recipients to induce cancer.
Some of the induced cancers start to grow and lead to the death of the host
while others regress. It has been possible to immunize animals with irradiated
tumor cells and suppress the growth of the cells injected thereafter indicating
that some immune surveillance against cancer exists 65 . Three major types o f
cells are important in the immune response to cancer. These cells are the
antigen-presenting cells, T-lymphocytes and natural killer cells.
2.2.1 Antigen-presenting cells (APC)
Antigen-presenting cells (APC) include dendritic cells, monocytes (Mo) and
macrophages (Mø). The function of AP cells is to engulf pathogens or dying
cells and to present their antigens for T cells. The antigens from the cellular
debris or pathogens must first be processed into peptide fragments. These
epitopes
are
then
presented
on
either
class
I
or
class
II
major
histocompatibility complex (MHC) proteins for T lymphocytes to recognize.
Dendritic cells are the strongest stimulators of the immune system 66 . They
have receptors for complement C3d, which improve the uptake of C3d-antigen
complexes to the cells for processing4. Dendritic cells have been used in
27
cancer therapy trials by treating isolated dendritic cells from the patient with
material extracted from cancer cells67, 68 . The hope is that after releasing
the modified dendritic cells back into the blood stream of the patient they
start an immune response by presenting the cancer antigens to the T cells.
2.2.2 T lymphocytes
T helper lymphocytes (Th; CD4+) and cytotoxic T-lymphocytes (T c ; CD8+) are
responsible for the generation of specific immune responses. Th cells release
cytokines that signal other immune cells to become activated. The cytokines
are released only if the T h lymphocyte recognizes an APC presenting with a
foreign peptide in its MHC as well as co-stimulatory molecules. These cytokines
can stimulate antibody production by B cells and a T c response. T c cells are
particularly important for eliminating those cells in our body that have been
infected with various viruses. T c cells are critical mediators
in tumor
immunology 68 . They also require the target antigen presented on MHC protein
plus co-stimulatory signals from T h cells to become activated. The t a r g e t
antigens on the tumor cells have been identified to be peptides from tumor cell
proteins. Although Tc cells can recognize tumor-associated antigen fragments
presented on MHC proteins, they usually do not become activated because
these TAAs are recognized as self proteins. Furthemore, cancer cells rarely
present the required co-stimulatory signals to activate Tc cells. The genetic
instability of tumor cells causes further problems. The malignant cells may lose
their tumor antigens by mutation, which lead to the appearance of escape
mutants that avoid the rejection. The tumor cells may also lose their MHC
molecules and thus become invisible for the cytotoxic T cells69. To further
suppress the activation of the immune system some tumor cells may even
start to express immunosupressive cytokines70 .
28
2.2.3 Natural killer cells (NK cells)
Another class of potential immune surveillance cells are NK cells that were first
discovered by their ability to identify and kill cancer cells. Unlike T and B
lymphocytes NK cells do not have specific receptors for a particular antigenic
target. NK cells recognize malignant or infected cells that have been coated
with antibodies by binding to the Fc region of the antibody with an Fc
receptor (FcγRIII or CD16) on their surface. CD16 recognizes IgG1 and IgG3
subclass antibodies. NK cells require certain cytokines to become activated
and for proliferation 71 . The destruction of the target cell is mediated by the
release of cytotoxic granules containing perforin and granzymes. The t a r g e t
cell dies by apoptosis and/or membrane damage. Furthemore, NK-cells can
discriminate normal cells from cells that do not express adequate amounts o f
MHC-I molecules72. NK cells express a variety of inhibitory receptors t h a t
recognize self-MHC class I molecules. These deliver an inhibitory signal to the
NK cell and prevent an attack against normal cells. They have also stimulatory
receptors that bind to a ligand on a target
cell72. The delicate balance
between opposite signals delivered by inhibitory receptors specific for MHC-I
molecules and the stimulatory natural cytotoxicity receptors (NCR; NKp46,
NKp30 and NKp44) regulate the effector functions of NK cells72. Only recently,
ligands for
another
stimulatory
receptor
NKG2D have been identified.
Interestingly, NKG2D-ligands (e.g. MIC and Rae1 proteins) are not expressed
by most normal cells but are strongly upregulated on transformed or infected
cells73. Diefenbach et al. have demonstrated that expression of the NKG2D
ligand Rae1 in several murine tumor cell lines resulted in a dramatic rejection
of tumor cells mediated by NK cells and/or CD8+ T cells74 .
29
2.2.4 Tumor antigens
The discrimination of cancer cells from normal ones is based on transformed
antigens or structures on the cell surface. Although cancer cells express only
few tumor-specific antigens that are unique to the tumor cells many cancers
express tumor-associated antigens (TAA) that can serve as potential targets
for immune response. TAA can be grouped into oncofetal Ag, tissue-specific
differentiation Ag and tumor-associated carbohydrate and glycolipid Ag. I t
has been shown that melanoma patients have T cells reactive with so called
MAGE antigens 75, 76 . The antigens of the MAGE family have a limited
distribution in
normal adult tissues. An exception is the testis that is an
immunologically privileged site. The most common melanoma antigens gp75,
gp100 or MART1 are overexpressed melanocyte differentiation antigens 75, 77 .
CA125 and epithelial cell adhesion molecule (Ep-Cam) are expressed by
ovarian and colorectal cancers, respectively. The 791Tgp72 antigen has been
characterized to be overexpressed in tumors including colorectal, gastric and
ovarian carcinomas and osteosarcomas. An overexpression of the 791Tgp72
antigen indicates a poor prognosis in colorectal cancer patients 78, 79 . In
subsequent studies, the 791Tgp72 antigen showed 100% sequence identity
with the complement regulator CD5580 . Another tumor marker that was first
isolated from the urines of bladder cancer patients and is currently used in
diagnosticks (BTA-TRAK test) was later found to be complement factor H81 .
2.2.5
Antibody responses
against
tumor-associated
antigens
in
cancer patients in vivo
Abnormally high density, mutagenic expression or changes in glycosylation can
make TAA immunogenic and lead to antibody production. Serological immune
responses to TAA has been detected in cancer patients. Often the antibodies
are directed against intracellular antigens (c-myb, c-myc, p21ras and p53)
that are released by necrotic tumor cells82. Breast cancer patients (43%)
30
and 32% of colon cancer patients have been found to be positive for anti-cmyb and p21ras antibodies, respectively83, 84. However, the detection of antip53 antibodies is usually associated with poor prognosis probably because
these intracellular antigens are not recognized by the immune system in live
cancer cells83. Instead, antibodies against plasma membrane antigens may
have an impact on tumor growth since they can react with live tumor cells.
The presence of some of these antibody/antigen complexes (PEM/MUC1) in
patients has been associated with a better prognosis 85 . Antibody responses
detected in cancer patients against cell surface antigens are listed in table 4.
Table 4. Antibody response against cell surface antigens in cancer patients82
Antigen
Origin of cancer
HER2/neu
Breast
PEM/MUC1
Ovary, breast, colorectal, pancreas
T, Tn, sialyl Tn
Breast, lung, pancreas
Gangliosides (GM1,
GM2, GD2)
Melanoma
2.2.6 Antibody-induced effector mechanisms
Antibodies are multifunctional proteins that have several effector functions
after they bind to their specific antigens. The IgG1 is the most widely used
antibody isotype in the therapy for cancer, because it activates
human
complement, recruits NK cells for ADCC, and has an extended half life in
plasma 86 . However, some studies suggest that IgA also recruits cytotoxic
cells in humans86.
Apoptosis is an important part of the cellular turnover. Cells that have to be
eliminated without inflammation enter apoptosis. Antibodies can kill nucleated
cells by inducing their apoptosis. Cross-linking of the Fas receptor (CD95) on
the target cell membrane by its ligand, FasL, or with anti-Fas antibodies,
induces apoptosis of the cells by activating of caspases 87 . Induction of Fas-
31
mediated apoptosis by anti-Fas antibodies has been demonstrated in solid
tumors implanted in mice. Unfortunately, treatment with anti-Fas antibodies o r
FasL causes severe damage to the liver87. Complement activation is likely t o
be required for efficient uptake of apoptotic
cells within the systemic
circulation. Exposure of phosphatidylserine on the apoptotic cell surface t o
serum has been shown responsible for complement activation and result in
coating the apoptotic cell surface with iC3b7. The binding of macrophage
receptors CR3 (CD11b/CD18) and CR4 (CD11c/CD18) with iC3b results in the
uptake of apoptotic cells7. Furthermore, C1q have been detected to bind
directly to
surface
blebs of
apoptotic
human keratinocytes,
vascular
endothelial cells and peripheral blood mononuclear cells19.
Antibody-dependent
cellular
cytotoxicity
(ADCC) occurs when an
effector cell binds to a target cell coated with antibodies and kills it. Cells
capable of ADCC include NK-cells, macrophages, neutrophils, eosinophils and
mast cells. ADCC has been studied most extensively in NK-cells. NK cells
express on their cell membranes CD16 molecules (FcγRIII) that bind IgG1 and
IgG3 on the target surface and thereafter release toxic substances into the
intervening space88.
Complement-dependent cytotoxicity (CDC) can lyse cells independently
of ADCC. In CDC the classical pathway is activated by IgM or IgG bound to the
tumor cell surface: subsequently, the formation of MAC causes the lysis o f
tumor cell targets. CDC is thought to be an important action mechanism o f
the anti-CD20 monoclonal antibody
treatment
that
is used therapeutically in the
of lymphoma patients. Malignant cells from B cell lymphoma
patients express CD46, CD55 and CD59 molecules at various levels resulting in
differences in the sensitivity of the cells to complement-mediated lysis. Lysis o f
cells from patients responding poorly to mAb therapy was increased 5- to 6fold after blocking both CD55 and CD5989 .
32
Complement-dependent
cellular
cytotoxicity
(CDCC) requires prior
activation of complement and deposition of C1q, C3b, iC3b or C4b on the
target cell. These ligands interact with the C1qR, CR1 or CR3 (CD11b/CD18)
receptors on NK-cells, polymorphonuclear leukocytes or macrophages t o
induce their cytotoxic activity90 .
Table 5. Antibody effector mechanisms in cell killing90
ADCC
CDC
CDCC
Initiator
IgG
IgG, IgM
C3b, C4b, iC3b, C1q
Transducer
FcγRI, II, III
C1q
CR1, CR3, C1qR
Effector
Mo, Mø, PMN, NK
MAC
Mo, Mø, PMN, NK
Abbreviations: ADCC, antibody-dependent cellular cytotoxicity; CDC, complementdependent cytotoxicity; CDCC, Complement-dependent cellular cytotoxicity; C1qR, C1q
receptor; CR1/CR3, complement receptor type 1 or 3; FcγR, Fc receptor for IgG;
iC3b, inactivated C3b; MAC, membrane attack complex of complement; Mo, monocyte;
Mø, macrophage; NK, natural killer cell; PMN, polymorphonuclear leukocyte.
2.2.7 Tumor antigens and immunotherapy of cancer
Ideally, tumor associated antigens (TAA) could be used as targets
for
monoclonal antibodies against malignant cells. B cell lymphomas have been
treated
successfully with anti-idiotypic
monoclonal antibodies that
have
become bound to the specific IgG-idiotype on the malignant cell surface. A
humanized mAb against CD20 (Rituximab®) is already in wide use for the
treatment
of
non-Hodgkin B cell lymphoma.
Rituximab was
the
first
monoclonal antibody officially registered for the treatment of cancer. The
mAb mediates complement-dependent cell lysis, antibody-dependent cellular
cytotoxicity and induces apoptosis 91, 92 . Trastuzumab, another humanized
monoclonal
antibody,
is
directed
against
the
HER-2/neu
receptor.
Trastuzumab has been shown to be most efficient in the treatment of HER-2overexpressing metastatic breast cancer. Combination of trastuzumab with
chemotherapy has produced higher response rates and longer survival than
treatment with chemotherapy alone93. This mAb, however, does not act by
activating complement but by blocking growth promoting activities of the
33
HER-2/neu receptor. Some tumor-associated antigens and their monoclonal
antibodies that are in clinical trials are listed in table 6.
Table 6. Non-conjugated monoclonal antibodies against tumor associated
antigens reviewed in94
Cancer
Antigen
IgG Type
Product
Ovarian carcinoma
CA125
mouse IgG
OvaRex®
Breast carcinoma
HER-2/neu
humanized IgG1
Herceptin®
Colorectal carcinoma
17-1A
mouse IgG2a
Panorex®
Chronic lymphatic
CD52
humanized IgG
Campath-1H®
CD20
humanized IgG
Mabthera®
Leukemia
Non-Hodgkin´s
lymphoma
(rituximab,IDECC2B8)
The major
problem
in active
immunotherapy
of
cancer
is the
poor
immunogenicity of cancer cells. Whole tumor cells have been used as vaccines
either mixed with adjuvants or after transfecting them with nonself proteins
or with immunomodulatory factors. However, isolated antigens that
are
selectively or abundantly expressed in cancer cells appear to function better
as vaccines than whole cells95. In experimental animals promising results have
been obtained by increasing the immunogenicity of cancer cells with nonself
peptides. Murine tumor lines with major histocompatibility complex (MHC)
class I-positive melanoma and colon carcinoma cells were injected into mice
together with MHC class I-matched peptide ligands of influenza virus. Mice
bearing live melanoma cells and colon carcinoma were efficiently cured by this
treatment 96 . T h cell responses have been elicited in patients with metastatic
melanoma
by subcutaneous injection of antigen-loaded (MAGE-3 tumor
peptides) monocyte-derived dendritic cells97.
In melanoma patients the
immunization with gangliosides produced an IgM response but no correlation
with antibody
titers
or
clinical outcome
was observed 82 . However,
in
melanoma patients immunized with GM2 and an adjuvant after surgery the
seroconversion correlated with increased survival98. Active immunotherapy
34
using cancer antigens (T and sialyl Tn) to induce antibody responses in
ovarian and breast cancer patients have produced IgM and IgG antibodies
with potential for CDC82 . Most of the breast cancer patients have responded
with IgM production and CDC to the the carbohydrate antigen globo H-keyhole
limpet
hemocyanin conjugate
vaccine admistered
together
with
QS-21
adjuvant 99 . A human anti-idiotypic antibody that mimics CD55 has been used
successfully in immunization of over 200 colorectal cancer and osteosarcoma
patients. 70% of patients showed CD55-specific immune responses with no
associated toxicity 100 . Although, natural antibody responses against cell
surface antigens have been detected in cancer patients (Table 4) only few
studies have indicated that complement is inherently activated in tumors in
vivo 101 . Sporadical deposits of C3 and C5b-9 have been detected in cervical
and breast cancer samples102, 103. This probably indicates a suppressing role
for the complement regulators.
Indeed, problems with the mAbs have been inefficient killing of the tumors as
well as inefficient penetration of the antibodies into solid tumors. To stimulate
tumor killing ability by mAbs toxic molecules like ricin 104 and Pseudomonas
toxin 105 have been coupled to antibodies. In other approaches mAbs have
been conjugated to chemotherapeutic substances such as adriamycin 106 o r
radioisotopes 107 in order to concentrate them to the tumor site 108 . Some
strategies have been designed to target and activate complement against
tumor
cells. These include heteroconjugates
antibodies and C3b or cobra
venom factor
composed
(CVF) that
of
monoclonal
activates
the
alternative pathway by replacing C3b in the C3 convertase 109, 110 . The
formed CVFBb complex is insensitive to complement inhibitors. In one study
interferon-treated
tumor
cells fixed C3b to
their surfaces through
the
alternative pathway111 .
Successful
complement
activation
on
tumor
cells may
have
multiple
consequences on the immune responses against the tumors. The cleavage
fragments
of
complement
proteins
C3a, C4a and
35
C5a
can
act
as
chemotaxins, leukocyte activators and anaphylatoxins to induce inflammation
in the tumor tissue. The larger fragments C3b, iC3b and C3d generated on the
target surface can interact with cell surface receptors of lymphocytes and
phagocytes to induce CDCC. There is evidence that tumor-cell bound C3
enhances the sensitivity of tumor cells to killing by activated macrophages 112 .
C3 deposition may also increase the antigenicity of the potential tumor
antigens on the tumor cells. Dempsey et al. have shown that fixing C3d into
hen egg lysozyme lowered the threshold level of B cells' response to lysozyme,
thereby increasing its immunogenicity4. Thus, C3d can act as a molecular
adjuvant and may be able to increase the immunogenicity of tumor antigens.
36
3. AIMS OF THE STUDY
The working hypothesis of this thesis was that complement could be used as
an effector mechanism to destroy malignant cells after membrane-bound
complement regulators have been inactivated on their cell surfaces.
Specifically, this study aimed at the following:
1. to analyze the significance of the membrane-bound complement regulator
CD59 for tumor cell survival from complement attack,
2. to determine the sequestration of CD59 into the various physicochemical
compartments of human breast milk,
3. to examine the ability of antibodies and complement components to
penetrate into microtumor spheroids and
4. to set up an animal model to test mAb-induced complement activation in
vivo.
37
4. MATERIALS AND METHODS
Methods and material used in the following studies I-V are described in detail
in the original publications and are listed in Tables 7-10.
Table 7. Antibodies used in studies I-V
Antibody
Description
Used in
512
mo mAb against rat Crry/p65
V
6D1
mo mAb against rat CD59
V
Anti-C1q
rb pAb against hu C1q
IV
Anti-C3
rb pAb against hu C3
IV
Anti-C3c
rb pAb against hu C3c
III
Anti-C3-FITC
go pAb against rat C3
V
Anti-C3bi
mo mAb against hu iC3b
IV
Anti-C5b-9
mo mAb against hu C5b-9
IV
BRIC216
mo mAb against hu CD55
I, III, IV
BRIC229
mo mAb against hu CD59
I, II, III, IV
BRIC230
mo mAb against hu CD55
III, IV
C1242
mo mAb against hu TAA: sialylated Tn III
C241
mo mAb against hu TAA: CA19-9
III
clone 528
mo mAb against hu HER2/c-erbB-2
I
IV
clone 3G8
mo mAb against hu FcγRIII
IV
clone 10.1
mo mAb against hu FcγRI
IV
clone C1KM5
mo mAb against hu FcγRII
GB24
mo mAb against hu CD46
I
J4.48
mo mAb against hu CD46
III, IV
Ma552
mo mAb against hu TAA: MUC-1
III
MG1, MG2, MG3,
MG4
mo mAb against CC531 cells
V
Ov185
mo mAb against hu TAA: CA125
III
Ov197
mo mAb against hu TAA: CA125
III
RDIII7
mo mAb against rat CD55
V
R2
rb antiserum against hu CD59
I
S2
rb antiserum against hu MCF-7
I, III, IV
YTH53.1
rt mAb against hu CD59
I, II, III, IV
Abbreviations: Ag, antigen; go, goat; hu, human; mo, mouse; mAb, monoclonal
antibody; pAb, polyclonal antibody; rb, rabbit; rt, rat; TAA, tumor associated
Ag
38
Table 8. Cell lines used in studies I-V
Cell lines
6D1
Description
mo hybridoma cell line producing
mAb to rt CD59
Caov-3
hu ovarian adenocarcinoma cell line
CC531
transplantable colon adenocarcinoma
of the Wistar derived Wag/Rij strain
MCF-7
hu breast carcinoma cell line
PA-1
hu ovarian teratocarcinoma cell line
SK-OV-3
hu ovarian adenocarcinoma cell line
SW626
hu ovarian adenocarcinoma cell line
T47D
hu breast carcinoma cell line
YTH531.1
rt hybridoma cell line (anti-CD59)
Abbreviations: hu, human; mo, mouse; rt, rat
Used in
V
III
V
I
III, IV
III
III
I, IV
I, II, III, IV
Table 9. In vivo tumor samples used in studies I, III and V
Cancer
Origin
Colon adenocarcinoma
rt liver
Ductal carcinoma
hu breast
Fibroadenoma
hu breast
Lobular carcinoma
hu breast
Mucinous cystadenoma
hu ovary
Mucinous cystadenocarcinoma hu ovary
Papillary cystadenocarcinoma hu ovary
Serous cystadenoma
hu ovary
Abbreviations: hu, human; rt, rat
39
Used in
V
I
I
I
III
III
III
III
Table 10. Laboratory methods used in studies I-V
Methods
Used in
Bichinonic acid (BCA) protein concentration
determination assay
I, II
Biotinylation of YTH53.1
III
Binding of CD59 to the TCC
I, II
CC531 tumor induction into rats
V
Cell damage visualization using propidium iodide
IV
Cell culture
I, III, IV, V
51
I, III, IV, V
Chromium ( Cr) lysis assay
Complement hemolysis assay
I, II
ELISA
V
I
F(ab´) 2 fragment preparation of YTH53.1 mAb
Flow-cytometry analysis (FACS)
III, V
Gel filtration
I
Immunoaffinity chromatography of CD59
I, II
Immunofluorescense microscopy
I, II, III, IV, V
Immunoblotting
I, II, III, V
Incorporation of CD59 into cell membranes
I, II
Induction of tumors in rats
V
I, II
Iodine (125I) labeling
Microtumor spheroid generation
IV
Milk fat globule (MFG) and MFG membrane isolation
II
Northern blotting
III
PI-PLC treatment
II, III
Plasma membrane isolation
V
RNA extraction
III
Scanning electron microscopy
IV
SDS-PAGE
I, II, III, V
Sucrose density gradient ultracentrifugation
I, II
Abbreviations: ELISA, enzyme-linked immuno-adsorbent assay; PI-PLC,
phosphatidylinositol-specific phospholipase C; SDS-PAGE, sodium dodecyl
sulfate polyacrylamide gel electrophoresis; TCC, terminal complment complex.
40
5. RESULTS AND DISCUSSION
The discovery of new tumor-associated antigens and developing mAb against
them is beginning to enable the use of complement as an effector mechanism
in cancer therapy.
Complement lysis of
cancer
cells is an important
therapeutic mechanism already in use in the treatment of certain types o f
lymphomas 89. However, mCRP on the surface of tumor cells reduce the
efficiency of CDC. Like on normal cells the GPI-anchored CD59 was found to be
expressed on malignant cells (I, III) and MFG (II). Differences in the expression
levels of CD59 and of the other mCRP were observed in tumor samples (I,
III)113 .
In vivo, the malignant cells usually grow
as
multicellular tumors
with
intercellular connections between tumor cells that may constitute a barrier
against complement attack. As shown in study IV they may prevent the
penetration of activated complement components into the tumor tissue.
The species selectivity of the activity of complement regulators requires an
homologous animal model for the studies of complement-mediated destruction
of cancer cells in vivo. To improve the efficiency of complement activity in
cancer therapy it is important to understand the complement regulatory
mechanisms of cancer cells. This thesis focused on studying the expression o f
mCRPs, and especially the significance of CD59 in preventing complementmediated lysis of breast
and ovarian cancer cells. Characteristically f o r
ovarian cancers, the malignant cells often remain confined to the abdominal
cavity and are in direct contact with ascitic fluid (AF) both as peritoneal
implants and as free-floating tumor cells. The individual malignant cells or small
cell clusters complicate the complete surgical removal of the tumor cells. The
hope is that
the remaining ovarian cancer cells could be removed by
intraperitoneal immunotherapy with mAbs.
41
5.1 mCRP on tumor cells and MFG (I, II, III, IV)
Immunofluorescence (IF) studies with mAbs against CD46, CD55 and CD59
showed that the complement inhibitors CD46 and CD59 were expressed on
plasma membranes of the breast cancer (MCF7, T47D) and the ovarian
cancer cell lines (Caov-3, PA-1, SK-OV-3 and SW626) studied (I, III). The
expression of CD59 was also confirmed by immunoblotting (I, III) and by
isolating the CD59 molecule from T47D and MCF7 cells (I). CD55 was detected
in an all examined cell lines except on PA-1 cells by IF. However, its expression
level was clearly lower than that
Complement regulator
obtained at
of the other complement regulators.
expression was also studied on tumor
samples
mastectomies and ovarectomies (I, III). CD46, CD55 and CD59
were detected in vivo in cryostat sections of solid breast tumors. A total o f
12 specimens (ductal carcinoma: 5 cases, metastases of ductal carcinoma: 2
cases, lobular carcinoma: 3 cases and fibroadenoma of the breast: 2 cases)
were examined by IF microscopy. CD59 was found to be strongly expressed
by all the tumors (I). Staining appeared significantly stronger in the tumor
cells than in the neighboring connective tissue. In cells that showed an epithelial
polarized pattern the strongest staining for CD59 was seen at the apical
membranes. A very strong staining for CD59 was seen within the ducts and
on cell surfaces adjacent to the duct lumen. In all tissues the endothelia o f
blood vessels appeared strongly positive for CD59.
Twentyeight ovarian tumor samples were obtained during ovarectomies ( 3
serous cystadenomas,
13
mucinous cystadenomas,
8
serous
papillary
cystadenocarcinomas and 4 mucinous cystadenocarcinomas). CD46 was
expressed on the epithelial cells of all the ovarian tumors.
CD55 was
expressed in 21 out of 28 cases. In general CD55 expression was relatively
weak. CD59 was strongly expressed on the epithelial cells of all the ovarian
adenomas and carcinomas. No apparent difference in staining intensity was
detected between adenomas and carcinomas (III). The distribution of CD59
expression on MTS grown from T47D cells was analogous to tumor samples
42
from
breast
cancers (I, IV). In some studies
the
expression
of
the
complement-regulatory proteins CD55, CD46 and CD59 has been found to be
deregulated in cancer, with tumors showing loss of one or more inhibitors and
strong overexpression of the others100 .
Since a strong expression of GPI-anchored CD59 was seen in breast duct
epithelia (I) its presence in human milk was also analyzed. Milk fat
is
composed primarily of triglycerides that are secreted into milk enveloped by a
membrane derived from the epithelial cells of the mammary gland. The milk f a t
glubule membrane consists of three zones. The two
outer
layers are
separated from the core by a proteinaceous coat. The narrow middle layer is
an apparent plasma membrane while the outermost layer or glycocalyx is rich
in carbohydrates and corresponds to a typical glycocalyx of a cell114. The
MFG-membranes contain a variety of glycoproteins that have been used t o
raise antibodies able to
detect
surface antigens on malignant
human
mammary cells115 . Milk fat globules were isolated from human colostrum and
milk by successive centrifugation (II). In phase contrast
microscopy the
apparent MFGs in the washed cream layer appeared heterogeneous in size
and showed no internal structures. Immunostaining with the BRIC229 mAb
showed that the MFG particles were covered with the CD59-antigen. The
CD59-specific fluorescence was not homogeneous but appeared in clusters on
the surface of the MFG particles. Occasionally CD59-specific staining was seen
on aggregates
outside the MFG particles. The membranes of MFG are
unstable, and soon after secretion into alveolar lumen the MFG lose some o f
their membranous material through vesiculation116 . The inhomogeneous CD59containing aggregates outside the MFG-particles (II; Fig. 1C) may represent
material that has become shed off from the membranes. Recently, we
observed that
shedding of CD59 and CD46 in vesicles is a common
phenomenon of ovarian and breast cancer cells117 .
43
5.1.2 Characterization of CD59 on T47D cells and MFG (I, II)
CD59 from breast cancer cells (I) and MFG particles (II) was purified t o
further characterize it. The MCF7 and T47D cell membrane-extracts were
solubilized with 60
mM n-octyl-ß-D-glucopyranoside.
The extracts
were
subjected to YTH53.1 anti-CD59 affinity column and the eluted material was
analyzed by SDS-PAGE and immunoblotting. The isolated proteins migrated as
broad bands with apparent molecular weights ranging from 19 to 25 kDa (I).
The pattern was similar to that of CD59 purified from erythrocyte
cell
membranes (CD59E) although, depending on the amount of sample loaded in
the gel, the smears of CD59E sometimes extended to 30 kDa (not shown).
The smear for soluble CD59 isolated from human urine (CD59 U) started from
a slightly higher Mw (molecular weight; 21 kDa) than that of the lipid-anchored
CD59. Additional 28 and 32 kDa bands in the immunoblots were apparently
due to nonspecific reactivity of the samples with the secondary antibodies,
because these bands were seen also in the controls where the primary
antibodies
were
omitted.
Purified
radiolabeled
T47D-CD59
became
incorporated into the rabbit erythrocytes indicating that the isolated T47DCD59 had not lost its glycophospholipid anchor. Under similar conditions
urinary CD59 did not become associated with the erythrocytes.
CD59 was affinity-purified in the same manner from milk and MFG-membranes
(CD59 M ) and analyzed by SDS-PAGE and immunoblotting using the BRIC229
mAb (II). In the CD59M preparation the BRIC229 anti-CD59 mAb bound
reproducibly to discrete bands with molecular weights ranging from 19 to 2 3
kDa. The pattern of distinct bands, usually three or four in number, was seen
in all MFG samples examined. Control CD59E was visible as a diffuse smear
from 18 to about 28 kDa. CD59M resembled CD59 isolated from MCF7 breast
carcinoma cells that also had distinct bands in the immunoblot. In SDS-PAGE
the affinity-purified CD59M migrated as bands with molecular weights of 19,
20, 22 and 23 kDa. When CD59M was treated with endoglycosidase F its Mw
decreased to 14 to 16 kDa when analyzed in a 15 % SDS-PAGE slab gel under
44
reducing conditions. Heterogeneity in the Mw of CD59 is due to variable
branching and sialylation of the N-linked oligosaccharide side chain, which is
linked to the Asn 18 residue in the polypeptide chain (Fig. 2) and constitutes
about 25 % of the molecular mass of CD59118 .
The affinity-purified T47D-CD59 and CD59M retained their functional activity as
judged by inhibition of complement lysis of guinea pig erythrocytes (GPE) when
incorporated
into their
membranes.
GPE activate
complement
via the
alternative pathway leading to deposition of MAC on the cell membranes and
eventually cell lysis. CD59 inhibits lysis by binding to the nascent C5b-8-complex
and preventing the insertion and polymerization of C9 45, 46 . When CD59 is
mixed with GPE it spontaneously incorporates into the cell membrane via its
hydrophobic phospholipid anchor. The full activity of CD59 requires the
presence of its glycolipid tail as the soluble molecule is approximately a 200fold
less efficient
inhibitor
of
cell lysis than
the
lipid-tailed CD59119.
Incorporation of T47D-CD59 into GPE was found to lead to inhibition of lysis o f
the cells by NHS. The functional activity of T47D-CD59 was equivalent to t h a t
of CD59E (I). The C-lysis inhibitory effect of incorporated T47D-CD59 could be
blocked by F(ab')2 fragments of the YTH53.1 mAb. When 125I-labeled CD59M
was incubated with GPE 29% of the radioactivity became incorporated into
the cells and remained there after repeated cycles of washing (II). Under
similar conditions only 4% of CD59U became associated with the cells.
Furthermore, the lysis of GPE by NHS could be inhibited in a dose-dependent
fashion by associating of different amounts of CD59M on their membranes. Full
inhibition of GPE lysis by 4% NHS started to occur at a CD59M concentration
of approximately 3 µg/ml (II: Fig. 4). The inhibitory activity of CD59E was
similar to that of CD59M giving a 73% inhibition at a concentration of 5 µg/ml.
Functional activities of T47D-CD59 and CD59M were tested
further
by
examining whether they bind to the terminal complement complexes (TCC). In
a TCC-binding assay complement is activated with inulin in C9-deficient serum.
Functionally active 125I-labeled CD59 binds to the formed SC5b-8 complex.
45
The bound CD59 can be separated
from inactive CD59 by high speed
centrifugation in a sucrose gradient. The 125I-labeled CD59M and T47D-CD59
bound to the soluble SC5b-8 complexes similarly as has been shown earlier f o r
CD59E45 and soluble CD59U53 (I).
It has been shown that melanoma cells constitutively release a functionally
active form of CD59 that contains an anchor and is able to insert into cell
membranes of homologous cells transiently increasing their expression o f
CD59 120. A soluble form of CD59, that retains its anchoring ability and
functional properties,
has been identified in body
fluids and in culture
supernatants of different malignant cells121. The physiological significance o f
CD59 in body fluids or as well in MFG remains unknown. The latter could simply
represent a GPI-anchored protein that has become sloughed off from the cell
membranes during lipid secretion. An intrinsic property of milk in killing and
preventing the invasion of pathogens in the gastrointestinal tract of the child
is accomplished mainly by noninflammatory mechanisms, i.e. by lactoferrin,
lysozyme,
fibronectin
and
IgA 122 .
Although,
the
components
of
the
complement system and complement-activating immunoglobulins IgG and IgM
are present in milk at low levels123, deposition of C3 fragments has been
detected on bacteria
incubated with milk124. IgA, the most
abundant
immunoglobulin in milk, does not activate complement and thereby also limits
inflammatory responses in the gut 125 . In addition, a number of antiinflammatory substances including antioxidants, catalase and histaminase are
present in milk125. This may be particularly relevant since during the post
partum period both the child and the mother are exceptionally prone t o
infections with associated inflammation.
5.2 Complement-mediated killing of MCF7 and T47D cells (I)
The potential effector mechanims of monoclonal antibodies in cancer therapy
include targeting of various toxic molecules, ADCC, CDC or CDCC. mAb have
46
been used in the therapy of lymphomas and melanomas with a limited
success 119, 126 . In the latter study intravenously administered monoclonal
anti-GD3-ganglioside mAb was observed to
become deposited
on solid
melanoma tumors and lead to lymphocyte and mast cell infiltration as well as
to local complement deposition. These results give hope that it is possible t o
launch an in vivo complement attack
against tumor
cells recognized by
antibodies.
Effective killing of the tumor cells requires their resistance to complement must
be overcome. In study (I) the cytotoxic effect of human complement against
MCF7 and T47D was examined in the presence and absence CD59-neutralizing
antibodies. When MCF7 cells were treated with F(ab') 2 fragments of the
YTH53.1 mAb prior to sensitization with complement activating antibody (S2)
an increase in antibody-induced complement-mediated killing of the cells was
observed (I). Approximately 12 µg/ml of the YTH53.1 mAb was required t o
obtain full neutralization of the tumor-cell CD59 activity. In the absence of the
sensitizing rabbit antibodies no lysis occurred with the YTH53.1 F(ab') 2 alone.
Also, in separate experiments the F(ab') 2 fragments did not induce lysis o f
human erythrocytes
antibody (rat
by human complement, whereas the whole parent
IgG2b), apparently by activating the classical complement
pathway, lysed human erythrocytes in the presence of complement. Using a
specific anti-CD59 mAb and F(ab') 2 fragments thereof it was possible t o
inactivate the functional activity of CD59 on cultured breast cancer cells. In
complement-mediated cell lysis experiments the maximum lysis of MCF7 cells
was 30 - 60% depending on the conditions used.
A polyclonal rabbit antibody S2 was found most effective in sensitizing the
T47D and MCF7 cells to complement lysis. A mAb against HER2/c-erbB-2
epidermal growth factor receptor did not sensitize MCF7 or T47D cells
sufficiently to initiate a lytic complement attack. On the other hand, the rabbit
polyclonal antibody was an efficient inducer of complement lysis, which was
enhanced by the anti-CD59 mAb. For sensitization of breast cancer cells t o
complement lysis the choice of an appropriate antibody is thus a critical
47
factor. This became evident also in the later studies (III and V). A majority o f
the mAb produced are of murine origin and many of them are poor activators
of human complement, especially the IgG1 isotype. Humanization of mouse
monoclonal antibodies is expected to widen their use for human cancer
therapy. It is possible to produce humanized mAbs that are functional in
mediating the immune effector functions, consisting of ADCC and CDC127.
Lately it has been shown that the recombinant form of the anti-HER2 mAb
(trastuzumab, Herceptin) activates complement leading to C3 deposition and
complement-mediated lysis of breast
carcinoma
cells if the mCRP are
neutralized 128 . The fact that total lysis was not achieved using S2 and antiCD59 suggests that the cells have other protection mechanisms than CD59
expression and/or that a more effective complement treatment
protocol
(higher doses, longer incubation times) should be used. When studied by
indirect IF microscopy using a mouse mAb against DAF (BRIC216) or MCP
(GB24) both regulators were found to be expressed by MCF7 and T47D cells,
although the expression of DAF was relatively weak on T47D cells. The
expression of functionally active CD59 on breast cancer cells shows that i t
has a role in protecting the malignant cells against lysis by the homologous
complement system and strongly suggests a similar function for it in vivo. The
neutralization of CD59 selectively on tumor cells, while saving the "bystander"
cells, poses a problem which we have attempted to solve by targeting the
anti-CD59 mAb to the tumor cells with the help of the biotin-avidin system 129 .
Biotinylation of the anti-CD59 mAb converts it into a nonactivator of C 130 and
may
facilitate
its
use
as
an
adjuvant
antibody
in
tumor-specific
immunotherapy. As a nonactivator of complement the biotinylated anti-CD59
mAb probably does not cause significant damage to bystander cells, although
it retains its ability to neutralize CD59 on the surface of tumor cells when
attracted to their surfaces by avidin-conjugated tumor-specific mAb.
5.2.1 Expression of TAA on ovarian cancer cells (III)
A critical factor for considering CDC in anti-tumor therapy is the selection o f
an appropriate TAA and mAb against them. Therefore, the expression o f
48
several TAA in ovarian cancer cell lines was studied using flow-cytometric
analysis (FACS). The Caov-3, PA-1, SK-OV-3 and SW626 cell lines had each an
individual expression pattern for the CA19-9, CA125 and MUC-1 antigens. The
Tn antigen was not expressed by any of the cell lines studied. For the
expressed Ags the staining intensities were generally low, and the expression
patterns were heterogeneous. An exception was the SW626 cell line, which
showed very strong expression of CA19-9 on its surface. In a subsequent
complement-activation assay the SW626 cells exposed to the anti-CA19-9
(C241) mAb and NHS showed a strong C3 deposition on the their surface in
FACS analysis. The amount of C3 deposition was much lower on PA-1 than on
SW626 cells, apparently reflecting the difference in CA19-9 antigen density
between the two cell lines. Similarly, the anti-MUC-1 mAb (Ma552) was able t o
induce C3 deposition on SK-OV-3 cells but not on Caov-3 cells. Neither of the
mAbs (Ov185 and Ov197) against CA125 Ag were able to induce C3
deposition on the Caov-3 cells, although the mAbs bound to the cell surface.
Compared with the mAbs the S2 anti serum efficiently induced C3 deposition
on all cell lines studied.
5.2.2 CDC and ovarian cancer cells (III)
In the next set of experiments, CDC induced by C241 mAb against CA19-9 in
SW626 and PA-1 cells was studied. In a chromium lysis assay 10% of the
SW626 cells were killed when sensitized with the C241 mAb and exposed t o
NHS. The neutralization of CD59 using biotinylated YTH53.1 mAb increased
the complement-mediated killing to 30%. Unlike SW626 cells, PA-1 cells were
not lysed by C241 and NHS, even if the complement regulatory function o f
CD59 was blocked. However, the PA-1 cells exposed to S2 were highly
sensitive to CDC (64%), and almost all PA-1 cells were killed (89%) after
simultaneous blocking of CD59. High sensitivity of the PA-1 teratocarcinoma
cells to complement lysis was associated with a relatively low expression of all
the complement regulators examined. These observations illustrate the need
for finding complement-activating mAbs against antigens that
expressed on the ovarian malignant cells. Neutralization
49
are highly
of CD59 with
biotinylated YTH53.1 reduced the resistance of almost all the cell lines to CDC,
except of the SK-OV-3 cells that remained somewhat resistant.
5.3 CDC and microtumor spheroids (MTS; IV)
MTS are spherical aggregates of cells growing in culture. They resemble tumor
tissue with their intercellular connections representing an intermediate between
monolayer cell cultures and tumors in vivo131, 132. The size of MTS may range
from a few cells to visible structures of 1 - 2 mm in diameter until the growth
is limited by restricted diffusion of oxygen and nutrients. Spheroids have been
used for studying the penetration of antibodies and the effects of lymphokine
activated killer cells against tumor tissue 133, 134 . In previous studies we
observed that in traditional killing assays cells growing in aggregates were
notably more resistant to C-mediated cytotoxicity than cells in suspension1. In
a killing assay individual PA-1 cells were incubated with a complementactivating antibody (S2) and a CD59-neutralizing mAb (YTH53.1B). As a
result, 85 % of the cells were lysed during a 30 min exposure to complement,
whereas 64% of the PA-1 cells exposed to S2 activating antibody alone were
lysed. Under similar conditions the lysis of the PA-1 cells in the MTS exposed t o
S2, YTH53.1 and NHS was approximately 8%. In the control spheroids
exposed to S2 and NHS the lysis remained at 4%.
Experiments in study
IV allowed
further
analysis and
visualization
of
complement-mediated attack against three-dimensional tumor tissue. A pulsed
complement attack
targeting
against MTS grown from T47D and PA-1 cells with
antibodies (S2) and complement in the presence of a CD59
neutralizing antibody resulted in progressive cell membrane damage and cell
detachment on the outer layers of the MTS. Full lysis of cores of the spheroids
appeared to be restricted by an arrest of complement activation at the C3
level. To quantify CDC against the cells in the spheroids a chromium ( 51 Cr)
release assay was used. Individual T47D spheroids were incubated with 3 µCi
of 51 Cr for 12 hours in 100 µl of cell culture medium. Earlier studies by
50
autoradiography have demonstrated that an overnight incubation leads t o
penetration of 51 Cr throughout
the spheroids 134 . The mean cumulative
spontaneous release of chromium during a 24-hour incubation of spheroids in
the RPMI 1640 medium containing 10% heat-inactivated FCS was 13% ± 0.6%
of the total
radioactivity
(n=4). This was in correlation with the total
remaining activity (90 ± 1.2%) that was counted from each spheroid after
the 24-hour incubation. The spontaneous release of chromium was considered
as background and was subsequently subtracted from the further results. To
study complement-mediated killing of T47D spheroids the S2 antibody was
used for activation the classical pathway of complement and the biotinylated
YTH53.1 mAb (YTH53.1B) for neutralization of the MAC inhibitor CD59 on the
cells. A 24-hour incubation with antibodies and a single dose of NHS resulted in
the release of 16 ± 4% (IV) of the spheroid-bound radioactivity.
By prolonging the time of exposure and replacing the complement components
consumed it was possible to partially overcome the complement resistance o f
the ductal breast carcinoma (T47D) and ovarian teratocarcinoma
(PA-1)
spheroids. During the incubation, at 2, 4 and 8 hours the spheroids were
isolated gently by pipetting and placed into new tubes containing fresh NHS
and antibodies to replace the complement components and IgG consumed, the
cumulative 51Cr-release was measured during the assay at the same time
points. In the killing assay, there was a clear lag phase in the 51 Cr release
during the first two hours of incubation (IV). T47D spheroids exposed to S2,
YTH53.1B and NHS for two hours showed only 1% lysis. At 4 hours 5% lysis
was achieved. A more significant lysis was not detected until 8 hours o f
incubation (15 ± 0.5%; n = 4). The mean (± SD) cumulative release o f
chromium from the spheroids exposed to S2, YTH53.1B and NHS during 2 4
hours of incubation was 33% ± 2.3%, while 62 ± 0.45% of 51 Cr was counted
to remain in the spheroids. In the control spheroids incubated with S0, normal
rat IgG and NHS for 24 hours (with replacements) the mean cumulative
activity released was only 3 ± 0.96% (n = 4) more than the spontaneous
release during the same time. In the spheroids exposed to either S2 o r
51
YTH53.1B and NHS the mean cumulative 51Cr-releases were 4 ± 1.5% and 5
± 0.6%, respectively (n = 8). The exposure of single T47D cells in suspension
to S2 alone and NHS resulted in 15% lysis of cells in 30 min (I). The
inactivation of CD59 increased the lysis only 13%. Thus, it is possible that the
detachment of the cells makes them more vulnerable to complement. In some
studies it has been shown that detachment of cells from the matrix induces
apoptosis 135 that may lead to additional cell killing. Increasing the incubation
time of the T47D spheroids to 48 hours only marginally increased the level o f
spheroid lysis (IV). The increase in the number of complement replacements t o
a total of 11 at 4-hour intervals during 50 hours of incubation did not
significantly enhance the tumor cell lysis either and a plateau phase was
reached after 42 hours (IV). However, since the spontaneous release o f
51 chromium was 30% at 48 hours the assay may not have revealed the full
extent of cell damage during prolonged incubations. With the PA-1 spheroids
3% of lysis was observed at 1 hour while at 2 hours already 37% lysis was
achieved (data not shown). Because of a tendency of PA-1 spheroids t o
spontaneously leak chromium during a prolonged treatment the lysis could not
be followed for longer than four hours at which time 45% lysis was observed.
Following complement exposure the surfaces of the spheroids became clearly
less coherent showing swollen and detaching cells as judged by light
microscopy. Photographic images indicated that the volume of the spheroids
was decreased by 28% from 0.194 ± 0.063 to 0.140 ± 0.05 mm 3 (mean ±
SD; n=30)
during
the
24-hour
pulsed treatment
with
antibodies
and
complement. The volume of the control spheroids exposed to S0, normal r a t
IgG and NHS decreased by only 5% during the 24-hour incubation (residual
volume: 0.185 ± 0.013 mm 3 ). Scanning electron microscopic analysis after
the complement exposure showed a release of relatively large vesicles from
the cell surfaces and porous remnants of cell membranes after severe cell
damage (IV). No similar changes were detected in the control spheroids
incubated with NHS, S0 and normal rat IgG (IV). In these controls, the surface
of the microtumors remained smooth with a fine microvillar coating.
52
To visualize death of individual cells in the spheroids, some of the spheroids
treated for various times with S2, YTH53.1B and NHS were incubated for one
hour with propidium iodide (PI) before freezing them into liquid nitrogen.
Fluorescence microscopical
analysis of
cryostat
sections
of
the
PA-1
spheroids showed a 2 to 3-cell layer thick frontier of dying cells on the
microtumor surfaces after 2 hours of complement exposure (IV: Fig. 3).
Apparently, some of the cells had become detached from the spheroid
surface. The nuclei of the detached cells were visible in the propidium iodide
stained specimens. New cell layers became exposed to complement after the
dead cell layer had been peeled off during the process. The cell death thus
proceeded as a frontier but was restricted mainly to the first four layers o f
cells on the periphery of the spheroids after a 24-hour exposure. In the
control microtumors, incubated with S0, nonspecific rat IgG and NHS, no dead
cells were observed on the microtumor surfaces. For comparison, in the T47D
microtumors the rim of dead cells did not extend deeper than one or two cell
layers from
the surface after
two
hours
of
incubation
(IV:
Fig. 5).
Furthermore, the cells still remained firmly attached to the microtumor tissue.
Following eight hours of exposure to C, the apparently dead T47D cells on the
surface remained adherent to the spheroidal body. After
24 hours o f
incubation, the PI-staining revealed a large pool of damaged cells detaching
from the surfaces of the spheroids. The necrotic cells in the centers of the
largest spheroids became also stained by PI. In the control spheroids t h a t
were exposed to S0, normal rat IgG and NHS, only the necrotic cells in the
center became stained, but no killing of cells on the surface was detected. This
indicated that propidium iodide was able to penetrate into the center of the
spheroids through the intercellular spaces.
The detachment of cells from the spheroid surface may be due to down
regulation of adhesion receptors and/or proteases released from the dying o r
sublytically attacked tumor cells. The proteolytic enzymes may start
degrade the extracellular matrix
and proteins
required for
to
the cellular
adhesion. Activation of metalloproteinases and degradation of extracellular
matrix occur during various physiological and pathological processes. These
53
include
conditions
that
require
tissue
remodeling
such
as
tissue
morphogenesis, inflammation and wound healing. On the other hand, e.g. the
complement serine esterase C1s has been shown to degrade type I and type
II
collagens136
and
to
activate
the
zymogen
form
of
the
matrix
metalloproteinase 9 enzyme 137. The released proteases might also activate
or inactivate complement components, and thereby have an enhancing o r
inhibitory effect on complement killing of the microtumor cells. Leukocyte
matrix metalloproteinases have been shown to inactivate C1 inhibitor t h a t
inactivates the C1r and C1s proteinases 138, 139 . Metalloproteases may also
cleave the membrane-bound complement regulators. Recently, we observed
that functionally active CD46 is was released from the surfaces of cancer cells
into solution by a metalloproteinase 117 . Enzyme release thus occurs and may
affect both tumor cell adhesiveness and complement sensitivity.
5.3.1 The penetration of mAb and complement into MTS
Cryostat sections of the complement-treated spheroids were examined using
IF to analyze the penetration of mAb and complement components into MTS.
During a 2-hour incubation the YTH53.1 mAb penetrated into the spheroid
tissue 4 to 5 cell layers deep (IV; Fig. 4). Following an overnight exposure t o
YTH53.1 and NHS the T47D spheroids became totally infiltrated by the
YTH53.1 IgG. In an earlier study, mAbs were also found to be capable o f
penetrating
into MTS 140. It is likely that
the
delivery of
complement
components and mAb into the spheroids occurs by diffusion through the
intercellular spaces. However, BRIC 229, another mAb against CD59, did not
completely pass through the spheroids during the same time (data
not
shown). Because of stronger binding to target antigens, antibodies of higher
affinity are predicted to diffuse more slowly into the tumor. In some tumor cell
killing assays higher-affinity antibodies have demonstrated
an improved
performance in CDC. In contrast, the lower-affinity antibody have penetrated
better into MTS133 .
54
Somewhat surprisingly, although C1q has a relatively large size (m.w. 460
kDa) it was able to penetrate 2 to 3 cell layers deep during a 2-hour
exposure to YTH53.1 and NHS (IV: Fig. 4) and almost through the spheroids
during 24 hours. The binding of C1q to the Fc part of an antibody is
noncovalent 141 analogous to
binding of antibodies to
an antigen. The
penetration of S2 was slower compared with the infiltration of the YTH53.1
mAb. In two hours the S2 IgG was able to penetrate only through the first
two cell layers. After 24 hours, the S2 IgG penetrated deeper forming a
gradient towards but not until the core of the spheroids. In the controls,
incubated with rat IgG, rabbit preimmune IgG (S0) and NHS, no deposition o f
nonspecific rat IgG, C1q
or rabbit IgG could be detected. To exclude the
possibility that antibodies bound to Fc receptors we used 10.1, C1KM5 and
3G8 mAbs for staining of FcγRI, FcγRII and FcγRIII on T47D cells, respectively.
No binding of any of these mAbs to the T47D cells was detected.
Immunofluorescence analysis showed that C3 (stained with the anti-iC3b
antibody) and C5b-9 deposits remained peripheral in the spheroids and were
restricted to the outermost 1 - 2 cell layers after 2 hours and 2 - 4 cell layers
after 24 hours (IV: Fig. 5) of complement exposure. PI staining was in
accordance with the staining for C3 and C5b-9 revealing that the complementmediated killing of the cells was limited to the microtumor surface. In the
controls, where S0, nonspecific rat IgG and NHS were used no deposition o f
C3 or C5b-9 were detected after 24 hours of incubation. Accordingly, no
complement-mediated death of cells was seen on the surfaces of similarly
treated microtumors. The main reason for the lack of cellular death in the
deeper parts of MTS may be the consumption of active complement on the
outer layers of the spheroids.
Complement treatment did not completely disrupt the macroscopic structure
of MTS. The cores of the spheroids retained their integrity during incubation
with antibodies and complement for 24 hours. It was thus possible to kill
efficiently cells growing on the surface of the spheroids but cells deeper inside
were protected from complement lysis. The plating of the spheroids after 5 0
55
hours resulted in the adherence and spreading of the cells over the cell culture
plate surface indicating that the spheroids still contained viable cells. The
results indicate that the resistance of cancer cells growing in the cores of the
spheroids to complement-mediated injury results at least partly from the
restricted spreading of C3 activation into the spheroids. Unlike, C1q the
forming C3b binds covalently with a thioester bond to a nearby cell surface
during complement activation. While C1q can diffuse relatively freely through
the the spheroid the penetration of C3 is limited, first, by rapid activation and
covalent binding and, thereafter, by rapid inactivation of C3b. Restricted
penetration of C3 is thus not due to a limited diffusion but because of its
specific biochemical properties.
A likely reason for the restriction of complement activation on to the spheroid
surface in our model is an effective complement regulation at the C3 level. In
these studies, we did not attempt to neutralize CD46 or CD55 which both
downregulate
C3 activation
on cell surfaces. Because of
its
stronger
expression on the spheroids 1 CD46 is likely to promote inactivation of both
C4b and C3b that have become bound to the tumor cell surfaces. Thus, CD46
could effectively block C3 convertase function in both the classical and the
alternative pathways. In our unpublished experiments the inactivation of CD46
together with CD59 (using GB24 and YTH53.1 mAbs, respectively) resulted in
60% lysis of the T47D spheroids.
5.4 A rat model to study complement activation in vivo (V)
The species-selective activity of complement inhibitors has been a hindrance t o
investigating the role of membrane-bound complement inhibitors in rodent
models of human cancer. It has been shown that human cancer cell lines are
significantly more sensitive to lysis by rat
complement than by human
complement 142. Regression of tumors has been observed in nude mice or r a t s
xenografted with human tumor tissue143-145 . In the study V we set up a r a t
model to analyze targeted complement activation in vivo. We characterized
56
the expression levels of complement regulatory proteins in a rat colon tumor
cell line (CC531) and the ability of tumor cell binding monoclonal antibodies
(MG1, MG2, MG3,and MG42a) to activate complement on these cells. MG42a is a
recombinant, complement-activating form of MG4. Rats were inoculated
subcapsularly into the liver with the in vitro growing CC531 cancer cell line t h a t
has been chemically induced in the same inbred strain of Wag Rij rats. The
inoculation resulted in tumor growth in the liver providing a reproducible
homologous animal tumor model.
5.4.1 CDC and rat colorectal cancer cells
Like in studies with human cancer cells (I) the flow cytometric analysis o f
expression of rat mCRP on the CC531 cells suggested that the rat colorectal
cancer cells are efficiently protected from complement activation. The CC531
cells expressed CD55 and CD59 analogously to human mCRP, plus Crry/p65
an additional rat complement inhibitor. As expected, CD46 was not detected
on CC531 cells. In rat, CD46 is mostly expressed in testicular cells. The
expression of Crry/p65, CD55 and CD59 on CC531 cells was confirmed by
Western blotting. Immunoblotting of the CC531 cell membranes with the 5I2
mAb, showed two bands of 55 and 65 kDa for Crry/p65. Also two bands, o f
65 and 185 kDa, were detected with the RdIII7 mAb (anti-CD55), as has been
previously described for CD5558, 146 . The anti-CD59 (6D1) mAb revealed one
band of 20 kDa.
5.4.2 C3 deposition on CC531 cells and CDC
To choose an antibody for in vivo studies the ability of the MG1, MG2, MG3
and MG42a mAb to activate the classical pathway of the complement system
was examined by a C3-ELISA. All mAb were able to activate the complement
system in this assay, although MG3 was a weaker complement activator than
the other mAbs. However, MG1 and MG2 were not
able to
activate
complement on the CC531 cells. Activation of the classical pathway
57
of
complement requires an adequate antigen density on the target cells. C1q has
to be bound by at least two IgG molecules for the activation to occur.
Inactivation of Crry/p65 with anti-Crry/p65 F(ab) 2 fragments increased C3
deposition three-fold compared to situation where only MG42a was used.
Inactivation of Crry/p65 and CD55 resulted in a synergistic increase in C3
deposition. As expected, the anti-CD59 mAb (6D1) did not have any effect on
C3 deposition. Because of their low complement activation potential on CC531
cells MG1, MG2 and MG3 were excluded from further studies. For the
complement lysis experiments MG42a was used to specifically activate r a t
complement on the CC531 cells and anti-mCRP mAbs to inactivate the
complement regulators on the cells. Activation of complement by MG42a mAb
and blocking of Crry/p65 and CD59 resulted in 55 % lysis of cells. Blocking o f
Crry/p65, CD55 and CD59 did not further increase the lysis. The control
antibody MG4 did not
induce complement lysis. In the presence of a
heterologous complement source, (baby rabbit complement; BRC) 83% Cmediated lysis was observed.
5.4.3 Complement activation in situ
To investigate the ability of MG42a to activate the complement system in situ
tumor tissue sections of untreated rats were incubated with either MG42a o r
MG4 mAb. Normal rat serum (NRS) was used as a source of complement. The
binding of MG42a mAb and deposition of C3 were analyzed by IF microscopy.
Both MG42a and MG4 bound to the tumor cells but only the recombinant
MG42a mAb was able to activate complement on the tumor sections. A clear
tumor cell membrane staining pattern was observed for both MG42a and MG4
mAb. In cells treated with MG42a a membranous-like staining pattern for C3
was observed. The MG42a mAb staining colocalized with that of C3.
58
5.4.4 Homing of mAb into tumors
and complement activation
in
vivo
Hagenaars et al. recently described that MG1, MG2 and MG4 were able t o
home to the tumor cells when injected intraperitoneally into tumor bearing
rats 147 . MG3 could neither be demonstrated on tumor cells when injected into
rats nor when incubated on tumor sections. Although the MG3 antigen was
detected by FACS on cultured CC531 cells it was not detected in the
immunoblots of isolated CC531 cell membranes (unpublished results). This
suggested that the antigen for MG3 mAb is not a protein or that its epitope
is lost during membrane processing. To determine if MG42a had a homing
ability comparable to that of MG4, the two mAb were injected into tumor
bearing rats. Tumors were grown for five days and rats were injected with
MG42a, MG4 or PBS. Two days after the mAb injection the tumors were
removed and snap-frozen. MG42a and MG4 were detected in cryostat sections
of CC531 tumors by using immunofluorescence microscopy. However, C3
deposition on tumor cells could
not be detected in MG42a or MG4-treated
rats.
The above experiments suggested that complement C3 may not become
deposited or becomes lost from CC531 cell tumors in vivo. The situation may
be similar as in the spheroids where the penetration of C3 into the MTS
remained peripheral (IV). In the experiments with spheroids the tumor cells are
in direct contact with complement compared to situation in vivo where the
complement components first have to cross endothelial cells of blood vessels.
This may restrict complement activation in the in vivo tumor.
In human cervical carcinoma sporadical deposits of C3d and C5b-9 have been
detected on the tissue sections. Furthermore, the C3d deposition was in
association with low expression of CD46 on the tumor cells103 . Interestingly, in
the same study it was observed that both C3d and C5b-9 were stained with
59
high intensity on blood vessels of tumor samples. In CC531 cells both CD55
and Crry/p65 are expressed (V). Complement activation in the rat tumor
interior can be restricted by CD55 or Crry/p65 or the surface layers of the
C3b/iC3b-bearing tumor
cells may
become
cleared
away
by
the
rat
phagocytes. These findings support the idea that complement activation on
tumors
is efficiently inhibited at
the
complement
convertase
level. As
mentioned earlier, CDC is thought to be an important effector mechanism in
the therapeutic treatment of some lymphomas 89, 92 . However, lymphoma
cells are in direct contact with serum complement and this together with
efficiently sensitizing mAb may overrun the C3-convertase inhibitors.
Taken together this study shows that mAb can reach tumors in vivo but
multiple mechanisms can restrict the access of complement components t o
the tumor cells.
60
6. CONCLUSIONS
The studies included in this thesis demonstrate that human breast (I) and
ovarian tumors (III) strongly express the complement membrane attack
inhibitor CD59, and to a lesser extent CD46 and CD55. With a mAb against
TAA (CA19-9) that is expressed by an ovarian cancer cell line (SW626) it was
possible to kill these cells using NHS as a source of complement (III).
Expression of CD59 apparently makes the tumor cells resistant to CDC, but
this resistance can be partially overcome by specific mAb that neutralize the
activity of CD59 (I, III). Study III indicated that the target antigen density on
the cancer cells is a critical factor for efficient CDC.
CD59 is expressed on the surfaces of normal and malignant cells. It was also
detected in the breast duct epithelial cells, from where it apparently became
shed to breast milk. Within the milk CD59 was present on the milk fat globule
membranes (II). Like on cell membranes CD59 in MFG had a glycolipid anchor
and maintained its functional activity.
Micrometastases and residual malignant cells after surgery constitute an
important
clinical problem
in cancer
therapy.
The approach
of
using
complement against tumors in vivo is currently limited by the shortage o f
tumor cell specific and complement activating antibodies. Many of the mAb
that are produced in murine hybridoma cell lines belong to the IgG1 subclass
that in general is a poor activator of human complement. Another problem is
the activity of complement regulators on the malignant cells. However, as
implied by the current and our earlier studies it is possible to specifically
target complement attack against tumors if suitable mAb are available. As
shown here, mAb (IV, V) and even large complement molecules, like C1q (IV),
can penetrate into the tumor tissue. Partial destruction of microtumors could
thus be achieved by neutralizing the mCRP and optimizing the antibodyinitiated complement attack. For further studies, the spheroid system offers a
useful model.
61
Like human cancer cells rat colorectal cancer cells also express complement
regulators on their membranes (V). It was possible to kill CC531 cells by
activating rat homologous complement with a specific mAb against these
cells. However, the homologous tumors
transplanted
into the rat
were
resistant to complement activation in vivo although the activating mAb homed
into the tumors. This indicates that the homologous complement inhibitors still
pose a difficult obstacle for mAb-therapy of tumors in vivo. However, by
engineering suitable mCRP-neutralizing antibodies and selecting appropriate
tumor-specific
C-activating mAbs it is, in principle, possible to
complement attack against malignant cells.
62
target
7. ACKNOWLEDGMENTS
The thesis was carried
out
at
the
Department
of
Bacteriology
and
Immunology, Haartman Institute, University of Helsinki. I am grateful to the
present and former heads of the department Professor Olli Mäkelä and Acting
Professors Seppo Meri and Risto Renkonen for providing excellent working
facilities.
I am grateful to all those who have been working with me during these years
and made my work possible. I want to express my deepest gratitude to my
supervisor Docent Seppo Meri for his enthusiasm and guidance and an
opportunity to learn much during this time. He has given me much freedom in
the
choice
of
scientific
topics
and
encouraged
me
to
develop
my
responsibilities in science.
I am grateful to my reviewers Professor Mikko Hurme and Docent Timo
Paavonen who gave me valuable criticism and comments for improving this
thesis.
I warmly thank all collaborators and coauthors: Line Bjørge and Roald Matre
from the University of Bergen, Norway, T Wahlström, Helsinki University Central
Hospital, as well as Kyra Gelderman, Martin Hagenaars, P.J.K Kuppen and Arko
Gorter, from the Leiden University, the Netherlands.
I am grateful to Docent Aaro Miettinen, MD, Riitta Väisänen, Maiju Solin, Anni
Haltia, Jukka Reivinen, Laura Kerosuo, they have helped and advised me
whenever needeed. Their lab is full of exciting gadgets and antibodies t h a t
have been helpful in performing the experiments. They have also made the
department a fun place to work at. I warmly thank Docent Pentti Kuusela. He
has always been helpful in sharing his knowledge. I am indebted to Docent
Matti Kaartinen who taught me methods in molecular genetics. I warmly thank
Mine Eray for sharing her knowledge in science. I also thank Docent Ilkka
63
Seppälä for his helpful advice. I thank Monica Schoultz, for technical assistance
with the flow cytometric analyses.
I warmly thank my previous and present colleagues. I am grateful and
priviliged for having had the opportunity to work with Timo Lehto, Jorma
Tissari, Antti Väkevä, and Sakari Jokiranta. I thank Taru Meri and Hanna
Jarva for brain-storming and for making work in the lab fun. I am indebted t o
Riina Rautemaa for her positive and friendly attitude and for our fruitful
discussions. I thank Sami Junnikkala for useful discussions and fun moments. I
want to thank our technician Marjatta Ahonen for her technical assistance
and precise work to making work in the lab more organized and easier. I
thank Mervi Närkiö-Mäkelä, Antti Lavikainen, Antti Harjunpää, Antti Alitalo,
Markus Lehtinen, Mikko Holmberg, Jinghui Yang, Zhu-Zhu Cheng, in our group
for the nice moments together. My special thanks go to Pauliina Luimula, and
Jyrki Eloranta, for
fun moments
outside
the
lab
and
Jyrki also
for
proofreading my thesis.
Finally, I am most grateful to my friends for making life outside the lab
pleasant and exciting. Special thanks belong to my parents for their guidance
and support.
I deeply acknowledge the financial and material support from the Sigrid
Jusélius Foundation, the Academy of Finland, Finnish Cultural Foundation, Maud
Kuistila Foundation, Finnish Cancer Society, Oskar Öflund Foundation, the
Haartman Institute, the Helsinki University Central Hospital, and the University
of Helsinki.
Helsinki, December 2002
64
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