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Revisión
Inmunología
Vol. 25 / Núm 3/ Julio-Septiembre 2006: 173-187
Control of complement activation by cancer cells and its
implications in antibody-mediated cancer immunotherapy
R. Pio
Division of Oncology (Center for Applied Medical Research) and Department of Biochemistry.
School of Medicine, University of Navarra, Pamplona, Spain.
CONTROL DE LA ACTIVACIÓN DEL COMPLEMENTO EN CÉLULAS MALIGNAS
Y SU IMPLICACIÓN EN LA INMUNOTERAPIA ANTITUMORAL CON ANTICUERPOS MONOCLONALES
Recibido: 10 Junio 2006
Aceptado: 5 Septiembre 2006
RESUMEN
El complemento es una parte esencial del sistema inmune
innato que participa en la eliminación de células extrañas al organismo. Debido al gran número de alteraciones genéticas y epigenéticas asociadas a la carcinogénesis, la transformación neoplásica puede incrementar la capacidad de la célula maligna para
activar el complemento. Este hecho está sustentado por estudios clínicos que demuestran una activación del complemento en
pacientes con cáncer. Sin embargo, las células malignas suelen
desarrollar mecanismos de protección que les hacen resistentes
al complemento. A la luz de recientes investigaciones sobre los
mecanismos de regulación de la actividad del complemento, el
papel de las proteínas inhibidoras, tanto de membrana como solubles, en la protección de las células neoplásicas es cada vez más
evidente. Esto podría limitar la eficacia de la inmunoterapia antitumoral basada en anticuerpos monoclonales que, entro otros
mecanismos, pueden activar el sistema del complemento. Se han
sugerido, y testado, distintas estrategias para la supresión de
los mecanismos de control de la activación del complemento.
En estudios in vitro e in vivo, la protección mediada por proteínas
reguladoras ha podido ser bloqueada mediante la inhibición de
su actividad o de su expresión por parte de la célula tumoral. También se han evaluado estrategias dirigidas a incrementar la capacidad de los anticuerpos para fijar el complemento o los mecanismos efectores asociados a su activación. Sin duda, un mejor
conocimiento del papel del complemento y de sus mecanismos
de control en cáncer ayudará al diseño de inmunoterapias antitumorales más eficaces.
PALABRAS CLAVE: Neoplasias / Proteínas inhibidoras del complemento / Inmunoterapia / Anticuerpos.
ABSTRACT
Complement is a central part of the innate immune system,
providing a highly effective means for destruction of non-self
cells. Given the numerous genetic and epigenetic changes associated with carcinogenesis, neoplastic transformation may be
accompanied by an increased capacity of the malignant cells to
activate complement. This is supported by clinical data that
demonstrate complement activation in cancer patients. However,
malignant cells are often resistant to complement activation by
the use of various protective mechanisms. In light of recent advances in the knowledge of the mechanisms regulating complement
activity, it begins to be clear that membrane-bound and soluble
complement inhibitory proteins play a key role in the protection of neoplastic cells from complement attack. This may hamper the clinical efficacy of cancer immunotherapy strategies based
on the use of monoclonal antibodies that, among other mechanisms, can activate complement. Some attempts have been already made to modulate antibody-mediated complement activation. In in vitro and in vivo studies, protection by complement regulatory proteins has been overcome by inhibiting their activities or
their expression by the target cells. Other strategies have been
aimed to increase the complement-fixing capacity of the therapeutic antibodies or to improve complement-mediated effector
mechanisms. Undoubtedly, a better understanding of the role
of complement and its mechanisms of control in malignant cells
would help to design more efficient complement-mediated cancer immunotherapies.
KEY WORDS: Neoplasms / Complement inactivator proteins /
Immunotherapy /Antibodies.
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CONTROL OF COMPLEMENT ACTIVATION BY CANCER CELLS AND ITS IMPLICATIONS IN ANTIBODY-MEDIATED ...
THE COMPLEMENT SYSTEM AND ITS REGULATION
Complement is a central part of the innate immune
system, providing a highly effective means for destruction
of invading microorganisms, clearance of immune complexes
and elimination of dead and apoptotic cells(1). The complement
system is one of the most ancient parts of the immune system,
present in evolution long before the development of adaptive
immunity, with which it co-operates(2-4). Complement can
enhance humoral immunity(5), modifies T cell immunity(6),
shapes the development of the natural antibody repertoire(7),
and regulates tolerance to nuclear self antigens(8,9).
Complement is composed of both circulating and
membrane-bound proteins and can be activated via three
distinct pathways: the classical, the alternative and the lectin
pathway. The contact of the first component with an activator
on the target cell (i.e. an immunoglobulin, an activating
surface or certain carbohydrate structures) leads to the
generation of a cascade of activations in a precise order
depending on the pathway that is activated. Complement
proteins can be either zymogens that become active enzymes
upon activation of complement, effectors, control proteins
or receptors. The three complement pathways share the
common step of activating the central component C3, and
differ in the mechanism of target recognition. The classical
pathway of complement is initiated by the binding of C1q
to Fc regions of antigen-bound immunoglobulins (IgG or
IgM). C1q together with C1r and C1s, two serine protease
proenzymes, constitute C1, the first component of the classical
pathway(10). Conformational changes in C1q lead to the
activation of C1r, which, in turn, activates C1s(11). The activation
of the C1q complex subsequently activates complement
through the cleavage of C4 and C2 to yield the classical
pathway C3 convertase (C4b2a) that is able to cleave C3.
The alternative pathway is the phylogenetically oldest one
and is triggered by low-level activation of C3 to C3b by
spontaneously hydrolyzed C3 and activated factor B(12,13).
C3b can attach to the target cell membrane, and bind to factor
B that is cleaved by factor D to form the alternative pathway
C3 convertase (C3bBb). The lectin pathway is activated
following the recognition and binding of mannose-binding
lectin (MBL) to repetitive carbohydrate patterns containing
mannose and N-acetylglucosamine residues on pathogen
surfaces (14,15). MBL forms a C1-like complex with MBLassociated serine proteases (MASP). Conformational changes
in MBL lead to the cleavage and activation of complement
components C4, C2, which continue activation as in the
classical pathway(11). In all three pathways, cleavage and
activation of C3 results in the deposition of C3b on the surface
of the target cell, leading to the activation of the C5–C9
components and the formation of the cytolytic membrane
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VOL. 25 NUM. 3/ 2006
attack complex (MAC) that binds to cell membranes, disrupts
the membrane’s integrity and facilitates cell lysis in a process
known as complement-dependent cytotoxicity (CDC). When
C3b is converted to iC3b, complement-dependent cellular
cytotoxicity (CDCC) links with antibody-dependent cellular
cytotoxicity (ADCC) through the interaction of iC3b with
complement receptor 3 (CR3; CD11b/CD18) on mononuclear
phagocytes(16), natural killer cells(17), and lymphocytes(18).
Eventually, the complement cascade of proteolytic enzymes
releases the anaphylatoxins C4a, C3a, and C5a. These small
bioactive peptides act as chemotaxins and leukocyte activators,
mediating important proinflammatory responses(19).
To protect host cells from bystander killing and as a
mechanism of regulation, activation of the complement
cascade is highly controlled by several regulatory proteins.
Control proteins regulate complement at three main levels:
they can inhibit the protease activity of the proteins involved
in the activation cascade, facilitate the decay and destruction
of convertases, and control the formation of the MAC. At
least six complement regulators can be found soluble in
plasma: C1 inhibitor, factor I, factor H, C4b-binding protein
(C4bp), vitronectin (S-protein) and clusterin (SP40,40). C1
inhibitor prevents the cleavage of C4 and C2(20). Factor I
cleaves and inactivates C4b and C3b (21). Factor H and
C4bp have a decay-accelerating activity for the alternative
and the classical C3 convertases, respectively(22,23). These two
proteins are also cofactors for factor I. Vitronectin and clusterin
inhibit the insertion of the MAC into the membrane(24,25).
Complement activation can be also controlled by membranebound complement regulatory proteins (mCRPs) such as
complement receptor type 1 (CR1, CD35), membrane cofactor
protein (MCP, CD46), decay-accelerating factor (DAF, CD55),
and CD59 (protectin). CR1 functions as a cofactor for factor
I and dissociates the C3 and C5 convertases(26,27). CR1 has a
restricted tissue distribution (erythrocytes, most leucocytes
and tissue macrophages). CD46 is ubiquitously expressed
except on erythrocytes and works like CR1 in acting as a
cofactor protein for factor I-mediated cleavage of C3b(28).
CD55 is a glycosyl phosphatidylinositol (GPI)-anchored
protein that inhibits the activation of C3 and C5 by preventing
the formation of new C3 and C5 convertases and accelerating
the decay of preformed convertases(29). Finally, CD59, also
a GPI-anchored protein, prevents the formation of the MAC
at the terminal step of the complement activation cascade(30,31).
Both CD55 and CD59 are expressed on the surface of virtually
all cell types. mCRPs are usually species-specific, protecting
the cells only from autologous or homologous complement(32).
Structurally, CR1, CD46 and CD55 are closely related to the
soluble regulatory proteins factor H and C4bp. They belong
to the RCA (regulators of complement activation) family of
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Figure 1. Structure characteristics of human mCRPs CR1, CD46, CD55,
CD59 and the soluble regulators factor H and C4bp. These regulators, except
for CD59, contain extracellular short consensus repeats (SCR) domains. SCR
domains, also known as sushi domains or complement control protein (CCP)
domains, are sixty amino acids long and have four invariant cysteine residues
forming two disulfide-bridges per domain. CD46 and CD55 contain four SCR
domains each, and factor H twenty. C4bp exists in several isoforms. In the
figure the major isoform is depicted, which consists of seven α-chains (with
eight SCR domains each) and one β-chain (with three SCR domains) linked
together in their C-terminal parts. CD59 does not contain SCR domains; its
extracellular domain consists of a seventy amino acid chain with five disulfide
bonds. Both CD55 and CD59 are glycophosphatidylinositol (GPI)-anchored
proteins, while CR1 and CD46 are inserted into the cell membrane through
their transmembrane domains.
genes and contain a variable number of short consensus
repeat (SCR) domains, each 60 amino acids long (Fig. 1).
These proteins, together with complement receptor type 2
(CR2) are encoded on the long arm of chromosome 1, in a
region referred to as RCA gene cluster(33,34).
COMPLEMENT ACTIVATION ON MALIGNANT CELLS
Carcinogenesis is a multistep process in which cancer
cells evolve from normal cells through the acquisition of
several sequential genetic and epigenetic abnormalities.
These abnormalities result in essential alterations in cell
physiology that dictates malignant growth(35). During the
evolution of a cancer, the sequential alterations produce
changes in cell morphology and result in the expression of
many neoantigens. For example, a change in glycosylation
phenotype is considered a hallmark of cancer cells(36). Warren
et al. found that cell surface carbohydrates of tumor cells
showed an altered structure when parent cells and virustransformed cells were compared(37). In addition, abnormalities
can also be found in the expression and structure of membranebound proteins. Hundreds of genes that are either inactive
in the normal tissue of origin or expressed at relatively
low levels are activated in cancer. These elements that
R. PIO
distinguish cancer cells from their normal counterparts may
well be recognized by the immune system(38). This is the
basis of the immune surveillance hypothesis, which proposes
that the immune system surveys the body for tumor-associated
antigens, eliminating many or most tumors(39). A corollary
to this hypothesis is that tumor cells in progressive cancers
develop active mechanisms to escape immune recognition
or resist immune attack. Although there is not irrefutable
evidence for the existence of an effective immune surveillance,
a wealth of published data support the role of the immune
system as a primary defense against neoplasia and the
importance of the protective mechanisms developed by the
tumors(39,40). In this sense, clinical studies have suggested
that the complement immune system is activated in patients
with cancer in response to the expression of tumor associated
antigens(41-43). In the sera of patients with neoplastic diseases
and in the membrane of the malignant cells an elevation in
complement activity or in levels of complement components
has been observed in many studies. Patients with ovarian
cancer showed elevated levels of C3a and soluble C5b-9 in
the intraperitoneal ascitic fluid, suggesting an activation
of complement on the ovarian cancer cells(44). Niculescu et
al. reported that C5b-9 deposits may be seen on cell membranes
of primary breast cancers(45). Yamakawa et al. detected
deposition of C3 and C5b-9 in neoplastic thyroid tissues,
which was confirmed later by the report of elevated C3d,
C4d, and C5 deposits on papillary thyroid carcinomas(46).
MBL complement activation pathway was significantly
increased in patients with colorectal cancer compared with
healthy persons(47), and MASP-2 concentration in serum
proved to be an independent prognostic marker with high
MASP-2 levels predicting recurrence and poor survival(48).
Deposition of C3 and C5b-9 was sporadically found on the
tumor cells and the surrounding stroma(49). In lung tumors,
immunohistochemical analysis revealed a minimal deposition
of C3b with an apparently lack of activation of the lytic
MAC(50). Elevated complement levels correlating with tumor
size were found in lung cancer patients(51). Complement
components C3c and C4 were significantly elevated in almost
all 96 patients with lung cancer when compared with the
levels in a control group(52). High complement hemolytic
activity and C3 levels were observed in serum from children
with neuroblastoma(53). Patients with carcinomas of the
digestive tract and with gliomas showed also increased
serum complement activity(54,55). In vivo alterations in the
classical pathway activation were described in patients with
chronic lymphatic leukemia (CLL)(56,57). Besides, a strong
positive correlation was found between the length of survival
of the patients with CLL and the initial activity of the classical
pathway of complement(58). In contrast to these results,
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COMPLEMENT REGULATORY PROTEINS IN CANCER
Figure 2. Protecting mechanisms for complement resistance of cancer cells.
Extracellular protectors limit the activation of complement proteins on the
surface of the target cells, mainly controlling C3 deposition and the assembling
of the membrane attack complex (MAC). These regulators include mCRPs,
soluble complement regulators such as factor H, ecto-proteases that can cleave
complement components deposited on the cell membrane, sialic acid residues
that can limit C3 deposition, and ecto-protein kinases capable of phosphorylating
several substrates essential for the formation of the MAC. There are also
important intracellular mechanisms that can induce the elimination of the
MAC by exo- and endocytosis. The present review is focused on mCRPs and
soluble regulators. A review of other significant complement resistance
mechanisms can be found in Jurianz et al.(60).
patients with advanced metastatic brain tumors had reduced
complement titers(55). Mangano et al. reported normal total
complement and C3 levels in patients with breast, gastric
and colorectal carcinomas, whereas levels in those patients
who displayed metastases fell below the normal range(59).
Although most in vivo observations support that many
cancers activate the autologous complement system, it is
also well-known that the efficiency of complement-mediated
tumor cytotoxicity is hampered by various protective
mechanisms(60). Figure 2 summarizes the mechanisms used
by tumor cells to resist CDC. Many of these resistance
mechanisms are also used by normal cells to avoid accidental
activation or bystander effects due to local activation of
complement. However, it is also reasonable to assume that
to prevent injury by activated complement, cancer cells
develop additional mechanisms to inhibit complement
activation. In vitro studies have shown that lung cancer cell
lines are extremely resistant to CDC, and this resistance is
much higher than that observed in normal cells such as
human nasal epithelium primary cell cultures(61,62). One of
the best understood protective mechanism used by cancer
cells is the over-expression of membrane-associated and/or
soluble complement regulatory proteins. Many current
hypotheses propose that expression of these proteins on the
neoplastic cell membrane protects tumors from complement
activation.
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Expression and role of mCRPs in tumors
Several studies have analyzed the expression of
complement regulators in primary tumors and cancer cell
lines. mCRPs, which serve as important regulators for
complement-mediated self-injury, are overexpressed on
many tumor cell types. This topic has been extensively
reviewed(63,64). With the exception of CR1, most cancers
express at least two if not three mCRPs. As noted by
Fishelson et al., it is surprising the large variation in mCRP
expression that can be observed among tumor types and
even among different specimens of the same tumor type.
In an illustrative study, the authors analyzed sixteen
metastatic melanoma lesions and observed that nine
expressed both CD46 and CD59, two had CD59 only,
one had CD46 only, and four had neither CD46 nor CD59(65).
The obvious consequence for a high expression of one or
more mCRPs by a tumor cell would be a higher resistance
to complement activation.
In melanoma cell lines, levels of cell membrane CD59
were found to regulate their sensitivity to homologous
CDC(66). An inverse correlation was found between CD59
expression and the extent of CDC when the cells were
sensitized with an anti-GD3 ganglioside monoclonal antibody
(mAb). Treatment of the cells with a neutralizing antibody
against CD59 or a phosphatidylinositol-specific phospholipase
C to remove CD59 from the cell membrane enhanced CDC(66).
In prostate tissue, no differences in CD46 expression were
observed between normal prostate epithelial cells and
malignant cells. However, expression of CD59 showed a
slight increase in the metastatic cases(67). Xu et al. previously
reported high CD59 levels in prostate tumors correlating
with disease progression, as measured by tumor stage,
Gleason grade or prostate-specific antigen levels(68). Jarvis
et al. demonstrated an upregulation of CD59 in metastatic
PC-3 and DU145 prostate cancer cell lines, which after
inhibition of CD59 with a neutralizing antibody showed
higher sensitivity to CDC(69). In a series of colorectal cancer
patients, immunohistochemical expression of CD59 was
identified as a marker of poor prognosis(70). Blocking of CD59
with anti-CD59 mAbs on colonic, prostate, breast and ovarian
carcinoma cells led to a dependent increase in CDC(71,72). On
the other hand, an overexpression of rat CD59 in human
neuroblastoma cells conferred resistance to killing by rat
complement in vitro and increased the capacity of tumor
growth in vivo(73). Overexpression of CD59 in melanoma
cells also increased the resistance of these cells to CDC(74).
With regard to the role of CD55 in complement activation,
Loberg et al. reported an increased expression of CD55 and
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INMUNOLOGÍA
its receptor CD97 in prostatic tumors compared to normal
tissue. Additionally, CD97 expression was associated
with the transition to the malignant phenotype(67). Inhibition
of CD55 in prostate tumor PC-3 cells using siRNA-mediated
knockdown expression resulted in a significant decrease in
overall tumor burden in a mouse model of metastasis(75).
Expression of CD55 and CD97 was also higher in medullary
thyroid carcinomas(76). Interestingly, CD97 mRNA expression
directly correlated with the histopathological stage of tumors,
showing higher levels in advanced stages. In colorectal
carcinoma, CD55 was seen as a marker for poor prognosis(77).
In contrast, Madjd et al. reported that loss of CD55 was
related to poor prognosis in breast cancer(78). High expression
of CD55 was significantly associated with low-grade lymph
node negativity and with good prognosis. Survival analysis
showed that CD55 overexpression was associated with a
more favorable outcome. Deficiency of the homologue of
human CD55 in mice significantly enhanced T cell responses
to active immunization(79), suggesting that over-expression
of CD55 in tumors may modulate tumor immunity mediated
by the adaptive response.
CD46 is found in most tumors and is perhaps the mCRP
with the least level of variation between tumors and normal
tissue. Nevertheless, it has been reported that breast tumors
expressed high levels of CD46 that correlated with tumor
grade and recurrence(80). Higher levels of CD46 in breast
cancer were associated with estrogen receptor-positive
samples, and lower levels with a loss of differentiation and
epidermal growth factor receptor positivity(81).
Soluble forms of the mCRPs have also been detected
in cancer patients; although it has to be pointed out that
these forms are not restricted to malignant diseases and
can also be found under normal conditions(82,83). Sera from
cancer patients contained soluble forms of CD46(84,85). The
stroma of breast, colorectal, lung, renal, ovarian, gastric
and cervical carcinomas also contained mCRPs(49,50,86-88).
Cell lines from different cancers are also known to release
soluble forms of the mCRPs(44,72,86,87,89-92). These forms are
able to bind to the tumor cell, and should be considered
in the resistance of tumor cells to complement activation.
In colorectal cancer, soluble CD55 was present in stool
specimens and was proposed as a diagnostic marker of
poor prognosis(93,94). Soluble forms of C1 inhibitor, CD59
and CD46, and the soluble inhibitor factor H, were present
at higher concentrations in intraperitoneal ascitic fluid
from ovarian cancer patients than in serum samples. The
presence of these regulators, together with the expression
of mCRPs may explain why ovarian malignant cells had
surface deposits of C1q and C3 activation products, but
not of C5b-9(44).
R. PIO
Expression and role of soluble complement regulators
in tumors
Less attention has been paid to the expression and
contribution of fluid-phase regulators in the control of
complement activation on cancer cells. However, it has been
suggested that some of these regulators are also important
in the resistance of tumor cells to complement activation
and CDC. This is the case for the soluble regulator factor
H(44,95-98). Factor H is a 150 kDa glycoprotein present in human
plasma which inhibits the formation and activity of the C3
convertase(22,23,99). Besides, alternative splicing of factor H
mRNA yields a 42 kDa protein, named factor H-like protein
1 (FHL-1), which shares the complement inhibitory activities
of factor H (100,101). Expression of factor H and/or FHL-1
has been described in primary tumors and cell lines from
different origins (97,102-107). In fact, a clinically approved
immunoassay for the detection of bladder cancer in urine
is based in the quantification of factor H(106,108). Obviously,
factor H expression may have consequences in the resistance
of cancer cells against complement. H2 glioblastoma cells
were exceptionally resistant to CDC but, although these
cells strongly expressed CD59, CD46 and CD55, a combined
neutralization of these molecules did not increase their
sensitivity to complement killing(97,109). It was later demonstrated
that H2 cells produced factor H and FHL-1 and were able
to bind them, promoting C3b cleavage. Anti-factor H mAbs
enhanced cell death, confirming that factor H (or FHL-1)
was involved in the complement resistance of this cancer
cell line(97). In SK-MEL-93-2, a human melanoma cell line,
factor H was the dominant factor regulating the inactivation
of cell-bound C3b and was involved in the control of the
classical pathway of complement(96). An anti-factor H antibody
also enhanced complement-mediated killing of Raji cells, a
cell line obtained from a Burkitt's lymphoma(110). Members
of the SIBLING family protected murine erythroleukemia,
and human myeloma and breast cancer cells from complement
attack, likely by sequestration of factor H to the cell surface(111,112).
Factor H and FHL-1 were highly expressed by ovarian
carcinomas and both proteins were abundantly present in
ascites from these tumors(107). Varsano et al. showed that
lung cancer cells were extremely resistant to CDC in vitro,
and this resistance was much higher than that observed in
normal cells such as human nasal epithelium primary cell
cultures(61,62). In their studies, they also showed that neutralizing
antibodies against CD46 and CD59 were not effective in
increasing the susceptibility to CDC(62), whereas the same
antibodies were very effective in facilitating CDC of normal
respiratory epithelial cells(61). These findings suggested that
lung cancer cells have other mechanisms to resist complement
activation in addition to CD46 and CD59. Our group has
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VOL. 25 NUM. 3/ 2006
TABLE I. Unconjugated therapeutic monoclonal antibodies approved for use in Oncology
Name (Tradename)
Company
Isotype
Target
Cancer indication*
Rituximab (Rituxan)
Roche and Genentech
Chimeric IgG1
CD20
NHL
Trastuzumab (Herceptin)
Roche and Genentech
Humanized IgG1
HER2/neu
Breast
Merk
Chimeric IgG1
EGFR
Colorectal
Roche and Genentech
Chimeric IgG1
VEGF
Colorectal and lung
Millenium and Ilex
Humanized IgG1
CD52
CLL
Cetuximab (Erbitux)
Bevacizumab (Avastin)
Alemtuzumab (Campath-1H)
*NHL, non-Hodgkin´s lymphoma. CLL, Chronic lymphocytic leukemia.
recently demonstrated that factor H was frequently expressed
in non-small cell lung cancer (NSCLC). Factor H was also
secreted to the extracellular milieu and was able to bind to
lung tumor cell surfaces, inhibiting the activation of
complement(98). Lung cancer is not the only cancer type in
which blocking of CD46 and CD55 does not make cancer
cells sensitive to complement(72,113). In these cases, it would
be interesting to determine whether soluble complement
regulators, such as factor H or others, secreted by the cancer
cells, can control complement activation at the level of C3
convertases. C4bp, the functional analogue of factor H in
the classical pathway, may be also a good candidate to
regulate complement activation on cancer cells at this level.
In cancer patients with various non-metastatic solid tumors,
the C4BP mean plasma levels were significantly higher than
those in the control group(114). C4bp was able to bind to SKOV-3, SW626 and Caov-3 ovarian adenocarcinoma cell lines,
which may lead to an increased control of the classical
pathway activation(115).
In relation to the other soluble complement regulatory
proteins, some reports have described the expression of C1
inhibitor by some primary tumors and cancer cells(44,92,105,116,117),
although this is not always the case(118). Clusterin expression
is also upregulated in cancer progression and tumor formation.
This protein is expressed in a wide variety of tissues and
it is implicated in many diverse physiological processes,
such as sperm maturation, lipid transportation, tissue
remodeling, membrane recycling, cell–cell and cell–substratum
interactions, stabilization of stressed proteins in a foldingcompetent state, promotion or inhibition of apoptosis,
and complement inhibition. Trougakos et al. have reviewed
the properties of clusterin and its implication in cancer(119).
The wide distribution of this protein, its diverse physiological
effects and the lack of specific studies on the implication of
clusterin in the resistance to complement activation in cancer
cells, allows only speculation on the importance of its presence
in the control of complement activation in tumors.
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THE ROLE OF COMPLEMENT IN CANCER
IMMUNOTHERAPY
Tumor-specific and tumor associated-antigens can be
used as targets for mAb immunotherapy. An increasing
number of mAbs are currently used as therapeutic drugs to
control tumor growth (Table I), and many others are currently
being tested in preclinical and clinical trials(120,121). mAbs
normally utilize a combination of mechanisms in directing
cytotoxic effects to a tumor cell. Some of these antibodies act
by blocking important cancer activities, but many of them
are also able to activate the immune system through ADCC
or CDC(122). Chimerized and humanized mouse mAbs containing
the human IgG1 Fc-region are examples of complement
activating mAbs(123). The Fc-regions of membrane-bound
antibodies interact with the heterooligomeric complex C1q
and activate the classical pathway. Successful complement
activation may have multiple consequences on the immune
response against tumors. Complement activation leads to
formation of the MAC, fosters opsonization, and releases
powerful proinflammatory anaphylatoxins. In addition, it
has been proposed that the effect of complement can synergize
with other antibody-mediated mechanisms of action important
for the efficacy of these antibodies. The release of the
anaphylatoxins results in the recruitment of effector cells,
such as natural killer cells, into the tumor. CR3 expressed by
phagocytes and natural killer cells can be manipulated in
such a way that it will trigger CR3-dependent cellular cytotoxicity
or enhance ADCC of iC3b-coated tumor cells in the present
of the yeast cell-wall β-glucan(123). In a syngeneic mouse model
of metastatic lymphoma, a mAb directed against the ganglioside
GD2 expressed in EL4 lymphoma cells caused both CDC and
complement-dependent enhancement of ADCC(124).
Rituximab, an anti-CD20 mAb, is used for the treatment
of malignant lymphoma(125). However, in some cases with
bulky mass and at stage IV, lymphoma cells become resistant
to rituximab treatment(126). How this resistance occurs has
not yet been clarified. Rituximab may exert its activity
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through different mechanisms: inhibition of proliferation,
induction of apoptosis, CDC, and ADCC(126). Both in vitro
and in vivo data suggest that CDC may be one of the most
important mechanisms of action. Rituximab was shown to
cure immunocompetent mice challenge with murine
lymphoma EL4 cells stably transfected with human CD20.
However, its efficacy was completely eliminated when
the same study was done in syngeneic knockout mice lacking
C1q(127). The efficacy of rituximab, as for any other cancer
treatment with mAbs, may be limited by the expression of
complement regulatory proteins by the target cells. No
differences in the expression of CD59 have been reported
between normal and malignant B cells, whereas CD55
expression was shown to be different among individual
patients with B-cell malignancy(128). Clinical responses to
rituximab could not be predicted by the level of CD55 or
CD59 in patients with chronic lymphocytic leukemia (CLL)
or B-cell non-Hodgkin lymphoma(129,130). However, in vitro
data suggest that there may exist an inverse correlation
between the expression of these regulators and the susceptibility
to CDC in B-cell lymphoma cell lines(131,132). A decrease in
susceptibility to CDC with rituximab was dependent on
CD55 expression and CD55 expression correlated with tumor
size in lymphoma cells from patients with non-Hodgkin
lymphoma(133). Knockdown of CD55 by siRNA overcame
resistance to CDC in fresh lymphoma cells treated in vitro
with rituximab(133). A similar result from the same authors
was obtained with siRNA of CD55 in SK-BR3 breast cancer
cell line treated with trastuzumab, a clinically-approved
mAb against the Her2/neu receptor(133). These data confirmed
previous studies with two other breast cancer cell lines, in
which the blocking of CD59 and CD55 reduced the resistance
of these cells to CDC after treatment with trastuzumab.
ENHANCING THE EFFICACY OF ANTIBODY-BASED
IMMUNOTHERAPY BY MODULATING COMPLEMENT
ACTIVATION
Clinical efficacy of mAb immunotherapy may be enhanced
by overcoming protection with complement inhibitors
and/or by improving complement-mediated effector
mechanisms. Table II summarizes some of the strategies
that have been proposed for that purpose.
The effectiveness of membrane regulators in protecting
tissues from complement injury has provided an impetus to
explore the therapeutic application of blocking mCRPs to
improve antitumor therapy with mAbs. Several strategies
have been developed and tested experimentally in vitro and
in animal models. Theoretically, the protective capacity of
mCRPs can be overcome by different ways(64): blockade of the
R. PIO
TABLE II. Strategies to enhance the efficacy of mAb-based
immunotherapy by modulating complement activation
To overcome protection capacity mediated by complement regulators
• Blocking the complement regulator activities
• Down-regulating the expression of the complement regulators
• Removing the complement regulators from the cell surfaces
To improve complement-mediated effector mechanisms
• Modifying mAb by isotype switching, genetic engineering
or conjugation
• Using mAbs as a mixture rather than as individual ones
• Selecting antigens with a strong capacity to activate complement
regulator activity; down-regulation of mCRP expression; or
removal of the regulator from the cell surface. Numerous
papers, as mentioned above and reviewed previously(64), have
demonstrated the efficacy of neutralizing antibodies to block
mCRPs and to improve the complement-mediated activity of
mAbs able to recognize cancer-associated antigens and fix
complement. However, the challenging problem is the targeting
of mCRP inhibitors selectively to malignant cells, avoiding
thei effects on normal tissue. Macor et al. have proposed the
use of a biotin-avidin system developed by Paganelli et al.(134)
to target selectively neutralizing antibodies raised against
complement regulators(135). The use of therapeutic bispecific
mAbs that target a tumor antigen and simultaneously block
a major complement regulatory protein has also been
suggested(136,137). These antibodies recognize tumor antigens
and mCRPs such as CD59 or CD55(138-140). The efficacy of this
strategy has been demonstrated in a syngeneic lung metastasis
model of rat colorectal cancer(141). An alternative strategy to
abolish the control by mCRPs is to reduce its expression on
cancer cells. Some cytokines may induce downregulation of
CD55 or CD59 and thus affect the effectiveness of immunotherapy
with mAbs. Blok et al. investigated the effect of ten cytokines
on the expression of CD46, CD55 and CD59 in human renal
tumor cell lines and proximal tubular epithelial cells(142). Three
out of ten cytokines tested (IL-1β, IL-4 and TGF-β1) induced
consistent downregulation of mCRP expression on all tumor
cell lines studied. IL-1β downregulated the expression of CD46
and CD59, IL-4 the expression of CD46, and TGF-β1 the
expression of both CD46 and CD55. Besides, changes in the
expression of CD55 and CD59 were associated with changes
in the amount of C3 deposited and the extent of CDC,
respectively(142). However, most cytokines either enhance or
have no effect on mCRP expression(91,143-149). Besides, the effect
of the cytokines on mCRP expression varies, even when the
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cell lines studied are derived from the same tumor type, making
the in vivo effect highly unpredictable. Another option would
be the use of chemotherapeutic agents or other compounds
capable to modulate the level of expression of mCRPs(150-152),
although to date highly effective reagents for this purpose are
still needed.
In vitro studies have tested the possibility to remove
CD55 and CD59 molecules from the membrane with GPIphospholipase C. Lysis of melanoma, lung carcinoma and
cervical carcinoma cells increased following treatment with
this enzyme(49,62,66). GPI-phospholipase C treatment increased
sensitivity of CD20-positive IM9 cells to rituximab-mediated
CDC(153). However, it is important to note that the treatment
is not specific for mCRPs and would remove other surface
molecules, complicating the interpretation of these studies.
mAb-mediated immunotherapy might be also enhanced
by improving complement-mediated effector mechanisms.
A way to boost complement activation is the modification
of the therapeutic antibodies by isotype switching, genetic
engineering or conjugation. Heteroconjugates composed of
antitumor antibodies and molecules such as cobra venom
factor (CVF), C3b or iC3b have been used. CVF activates
the complement system by forming stable C3/C5 convertases
in mammalian serum(154). Combination of CVF with antibodies
or F(ab)2 fragments directed against different tumor-associated
antigens had an additive effect on CDC(155,156). In an orthotopic
pancreatic cancer model using nude rats, CVF conjugated
to a mAb with tumor-binding properties was able to increase
in vivo tumor infiltration by natural killer cells and
macrophages(157). The complement-activating capacity of
the 17-1A mAb was enlarged by conjugating it to CVF or
C3b(136). Other authors have also used antibodies conjugated
to C3b or iC3b to increase lysis of tumor cells(95,158). Kennedy
et al. increased complement activity by the combined used
of ribuximab and the mAb 3E7, specific for iC3b. 3E7 enhanced
iC3b deposition in CD20-positive Raji and ARH-77 cells
treated with rituximab. Consequently, the potential of
rituximab and IF5 (another anti-CD20 mAb) to facilitate
CDC was substantially enhanced in the presence of 3E7(159).
Bispecific antibodies may also be engineered to recruit
complement effector functions(160). Finally, to promote cooperation between CDC and ADCC, Gelderman et al.
proposed the use of β-glucan to activate CR3 on effector
cells and induce CR3-dependent cellular cytotoxicity(123).
A low initiation of CDC by mAbs may be also related
to a low density of antigenic epitopes on the target cell
membrane. This reduces the chance for the IgG to form
dimers required for complement activation. Some data in
the literature support this concern. Sensitivity to ribuximabmediated CDC in leukemia cells freshly isolated from patients
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VOL. 25 NUM. 3/ 2006
correlated with the expression levels of CD20 (161). The
acquisition of resistance to rituximab was associated to a
decreased CD20 expression(162). The antitumor activities of
several murine mAbs to HER-2 overexpressed on tumor
cells were more effective in activating complement as a
mixture rather than as individual antibodies(163). Chimeric
mAbs cMOV18 and cMOV19 against two distinct epitopes
of the folate receptor were only effective in combination,
and neutralization of CD46 and CD59 markedly enhanced
the susceptibility of tumor cells to CDC of ovarian cancer
cells(135). The selective alteration of the glycosylation pattern
can also enhance the lytic potential of humanized IgG1 mAbs
without affecting the affinity and specificity of the antibody.
Glycosylation variants of the therapeutic Lewis Y-specific
humanized IgG1 antibody IGN311 were able to lyse cells
more potently than the parent antibody, which may overcome
problems with low density of antigenic epitopes and lower
the minimally effective clinical dose(164). The molecular
architecture of the antigens selected for immunotherapy
seems also essential for the proper induction of CDC(165,166).
CONCLUSIONS AND PERSPECTIVES
Given the numerous genetic and epigenetic changes
associated with carcinogenesis, it is clear that tumor cells
express many neoantigens that may be recognized by the
immune system. However, cancer cells can develop
mechanisms to avoid immune recognition or activation(38).
The elucidation of these mechanisms may provide ways to
improve cancer immunotherapy. Although there are many
reports that describe abnormalities in complement levels
in patients with cancer, the role of complement in immune
surveillance against cancer has not been unequivocally
demonstrated. The complement system of patients may be
activated indirectly by immune complexes, concomitant
infections, or substances generated inside the tumor mass(64).
We still need to characterize more potential tumor associated
antigens able to stimulate complement, and to better
understand the intricate mechanisms of tolerance. We will
need to explore biologically relevant animal models of
cancer with and without complement deficiencies to
investigate the contribution of complement in cancer
immunosurveillance and the role of complement regulators
in the complement-mediated elimination of tumor cells.
Since most complement regulators operate in a species
specific manner, syngeneic animal models are probably the
best option for this kind of studies.
Whether or not one accepts that cancer is normally
suppressed by continuous immunosurveillance, experimental
and clinical evidences clearly support the idea that the
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INMUNOLOGÍA
immune system can be mobilized to attack malignant
cells that express tumor-associated antigens. Many in vitro
and in vivo studies have shown that blocking the function
of regulatory proteins sensitizes resistant tumor cells to
complement. These results have led to the hypothesis that
inhibiting the function of complement regulatory proteins
expressed on tumor cells will enhance immunotherapy. At
least in animal models, this has proved to be true(167,168).
However, complement regulatory proteins are widely
expressed on normal cells, and targeting inhibitory molecules
to complement regulators expressed and/or bound to tumor
cells in vivo is still a technical challenge. Besides, additional
strategies used by tumor cells to prevent complement
activation have received much less attention than the
expression of mCRPs, even though their role in complement
resistance of tumor cells is probably also important. Each
tumor may be equipped with different mechanisms of cell
protection from complement attack. It is conceivable that a
concerted action against different protective mechanisms
will be required to achieve efficient antibody- and complementmediated cancer immunotherapy(72).
ACKNOWLEDGEMENTS
The author thanks Dr Santiago Rodriguez de Cordoba
for helping with figure 1 and for critical reading of the
manuscript. Work in the author’s laboratory has been
funded by «UTE project CIMA», 2004-2006 AACR-Cancer
Research and Prevention Foundation Career Development
Award in Translational Lung Cancer Research, Instituto
de Salud Carlos III: Red de Centros de Cáncer RTICCC
(C03/10), and Ministerio de Educación y Ciencia (SAF2005-01302).
DISCLOSURES
The author has no financial conflict of interest.
CORRESPONDENCE TO:
Ruben Pio
Division of Oncology
CIMA Building
Pio XII, 55
31008 Pamplona (Spain)
Email: [email protected].
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