How Does Complement Affect Hematological Malignancies: From Basic Mechanisms to Clinical Application

Abstract

Complement, as a central immune surveillance system, can be activated within seconds upon stimulation, thereby displaying multiple immune effector functions. However, in pathologic scenarios (like in tumor progression), activated complement can both display protective effects to control tumor development and passively promotes the tumor growth. Clinical investigations show that patients with several hematological malignancies often display abnormal level of specific complement components, which in turn modulates complement activation or deregulated cascade. In the past decades, complement-dependent cytotoxicity and complement-dependent cell-mediated phagocytosis were fully approved to display vital roles in monoclonal antibody-based immunotherapies, especially in therapies against hematological malignancies. However, tumor-mediated complement evasion presents a big challenge for such a therapy. This review aims to provide an integrative overview on the roles of the complement in tumor promotion, highlights complement mediated effects on antibody-based immunotherapy against distinct hematological tumors, hopefully provides a theoretical basis for the development of complement-based cancer targeted therapies.

Full text

How Does Complement Affect
Hematological Malignancies: From
Basic Mechanisms to Clinical
Application
Shanshan Luo
1†
, Moran Wang
1†
, Huafang Wang
1
, Desheng Hu
1,2
, Peter F. Zipfel
3,4
*
and Yu Hu
1
*
1
Institute of Hematology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan,
China,
2
Department of Integrated Traditional Chinese and Western Medicine, Union Hospital, Tongji Medical College,
Huazhong University of Science and Technology, Wuhan, China,
3
Department of Infection Biology, Leibniz Institute for
Natural Product Research and Infection Biology, Hans Knöll Institute, Jena, Germany,
4
Faculty of Biological Sciences,
Friedrich Schiller University, Jena, Germany
Complement, as a central immune surveillance system, can be activated within seconds
upon stimulation, thereby displaying multiple immune effector functions. However, in
pathologic scenarios (like in tumor progression), activated complement can both display
protective effects to control tumor development and passively promotes the tumor
growth. Clinical investigations show that patients with several hematological
malignancies often display abnormal level of specific complement components, which
in turn modulates complement activation or deregulated cascade. In the past decades,
complement-dependent cytotoxicity and complement-dependent cell-mediated
phagocytosis were fully approved to display vital roles in monoclonal antibody-based
immunotherapies, especially in therapies against hematological malignancies. However,
tumor-mediated complement evasion presents a big challenge for such a therapy. This
review aims to provide an integrative overview on the roles of the complement in tumor
promotion, highlights complement mediated effects on antibody-based immunotherapy
against distinct hematological tumors, hopefully provides a theoretical basis for the
development of complement-based cancer targeted therapies.
Keywords: complement, hematological malignancies, immune evasion, tumor progression, immunotherapy
INTRODUCTION
Complement was initially identified more than 100 years ago due to a result of its bactericidal
activity ‘complementary’to the action of immunoglobulins and the role in phagocytosis of cellular
debris (1,2). As an essential part of innate immunity and an evolutionary old system, complement is
highly conserved among a wide variety of species, emphasizing its importance in immune defense
throughout evolution (3). Complement-dependent cytotoxicity (CDC) and complement-dependent
cell-mediated phagocytosis (CDCP) were fully approved to display vital roles in maintaining
homeostasis, fighting against infection and even in monoclonal antibody-based immunotherapies,
Frontiers in Immunology | www.frontiersin.org October 2020 | Volume 11 | Article 5936101
Edited by:
Junji Xing,
Houston Methodist Research Institute,
United States
Reviewed by:
Lubka T. Roumenina,
INSERM U1138 Centre de Recherche
des Cordeliers (CRC), France
Yifan Wang,
University of Texas Southwestern
Medical Center, United States
*Correspondence:
Peter F. Zipfel
peter.zipfel@leibniz-hki.de
Yu Hu
dr_huyu@126.com
†
These authors have contributed
equally to this work
Specialty section:
This article was submitted to
Molecular Innate Immunity,
a section of the journal
Frontiers in Immunology
Received: 11 August 2020
Accepted: 02 October 2020
Published: 29 October 2020
Citation:
Luo S, Wang M, Wang H, Hu D,
Zipfel PF and Hu Y (2020) How Does
Complement Affect Hematological
Malignancies: From Basic
Mechanisms to Clinical Application.
Front. Immunol. 11:593610.
doi: 10.3389/fimmu.2020.593610
REVIEW
published: 29 October 2020
doi: 10.3389/fimmu.2020.593610

especially in therapies against hematological malignancies.
Dysfunction of each step of the whole cascade disrupts
homeostasis and ultimately leads to severe diseases, such as
tissue damage, autoimmune diseases, infection and tumor
progression (4). Clinical investigations show that patients with
hematological malignancies often display abnormal level of
complement components, which in turn modulates
complement activation.
Hematologic malignancies are complex groups of disorders,
which becomes more and more into people’sfocussincethey
are often diagnosed in clinic. Although the general cure rate of
hematologic malignancies has been greatly improved and some
types have even high cure rates now, great challenges still exist
due to the large number of disease subtypes and high
heterogeneity. According to the 2018 global cancer statistics,
the incidence and mortality of non-Hodgkin lymphoma (NHL)
ranks first in hematological neoplasms, followed by leukemia,
multiple myeloma (MM), and Hodgkin lymphoma (HL) (5).
The causes of the diseases are various, but the host immune
conditions and tumor virulence factors are the main
determinants which closely correlated with the disease state.
The management of hematologic malignancies has traditionally
relied on chemotherapy and/or radiotherapy regimens to
control the tumor progressionandprolongthelifespan(6).
However, over the past two decades, significant progress has
been made for the development of monoclonal antibody (mAb)
therapies for hematologic malignancies. For example, anti-
CD20 mAbs, mainly rituximab (RTX) or obinutuzumab,
combined with chemotherapy agents have been approved for
treatment of diffuse large B-cell lymphoma (DLBCL), follicular
lymphoma (FL) and chronic lymphocytic leukemia (CLL),
respectively (7,8). Complement-dependent cytotoxicity and
complement-dependent cell-mediated phagocytosis display
vital roles in mAb-based immunotherapies. However, tumor-
mediated complement evasion might be a big challenge for such
atherapy.
In this review, we will first summarize complement
activation, function, and regulation, present an overview of
the “double edged”roles of complement in tumor progression,
then provide a deep insight into the roles of complement in
hematological malignancies, and further discuss complement
mediated effects on antibody-based immunotherapy against
hematological tumors.
COMPLEMENT ACTIVATION, FUNCTION
AND REGULATION
The complement system comprises more than 60 different
components which include main components, various activation
products, effector components, regulators, and several surface
bound complement receptors. Soluble complement exists in the
body fluids, displaying multiple immune effector functions.
Complement activation occurs in a sequential manner via three
different pathways and consists of four main steps: initiation, C3
convertase formation and amplification, C5 convertase formation,
and the assembly of the terminal complement complex (TCC),
also known as membrane attack complex (MAC). The alternative
pathway (AP) is initiated spontaneously and constantly. The lectin
pathway (LP) is activated uponbinding of mannose-binding lectin
to mannan and carbohydrate structures on microbial surfaces. The
classical pathway (CP) is activated via antigen–antibody
complexes or by C-reactive protein (4,9). Activation of all three
pathways results in the generation of C3 convertases that cleave C3
into C3a and C3b, followed by C5 convertase formation that
cleaves C5 into C5a and C5b, and the generation of TCC (3,4)
(Figure 1).
The cleavage product C3b binds to target surfaces, where it
acts as opsonin and mediates recognition and phagocytosis by
host immune effector cells (10,11). C3a and C5a function as
anaphylatoxins which initiate inflammation. Furthermore, C3a
also has antimicrobial activity by binding to the cell surface of
microbes and induces membrane perturbations and release of
extracellular material (12). In addition, complement also
functions as a link between innate and adaptive immunity. C3
synthesis by myeloid cells, a relatively minor source of
complement, provides a critical function during the induction
of humoral B responses to peripheral herpes simplex virus
infection. Further, macrophages derived from bone marrow
produce sufficient C4 to restore the humoral response to virus
infection in C4-deficient animals, demonstrating local
complement C3 and C4 production are required to enable
efficient B cell responses (13,14). Immune responses of T cell
to Listeria monocytogens were impaired in the absence of C3
(15). Besides that, complement activation products C5a/C3a and
its receptors (C5aR/C3aR) have a clear role in directly and
indirectly promoting T cell activation and proliferation and as
such, promoting allograft rejection, autoimmunity, and fighting
infection (16–18).
Due to its potency and the damaging effects, many
complement components are engaged in regulation (Figure
1). Complement regulators function at all levels of the cascade
and are classified into two major classes: fluid phase regulators
and membrane-integral complement receptors, such as Factor
H, Factor H like protein 1 (FHL-1), C4b binding protein
(C4BP), C1 inhibitor (C1INH), complement Factor H related
protein 1 (CFHR1), CFHR2, CFHR3, CFHR4, CFHR5, as well
as complement receptor 1 (CR1), CR2, CR3, CR4, CD46,
CD55, CD59, CRIg, vitronectin, clusterin as well as
carboxypeptidase N (2,19,20). Being highly regulated by
these regulators, complement forms an important, central
immune defense line and mediates cell integrity and tissue
homeostasis. Additionally, beyond complement regulation,
several of the above-mentioned regulators have additional
activities, such as mediating cell adhesion and extracellular
matrix interaction, or linking the complement cascade with
other important physiological networks (e.g. the coagulation
cascade) (21). However, due to the complement dysfunction,
many disease pathologies including tumor progression, tissue
damage, autoimmune diseases and infection may take
place (22).
Luo et al. Hematological Malignancies and Complement
Frontiers in Immunology | www.frontiersin.org October 2020 | Volume 11 | Article 5936102

ROLE OF COMPLEMENT IN TUMOR
PROGRESSION: THE TWO SIDES
OF THE COIN
Complement Mediated Anti-Tumor Effects
The complement system is a double-edged sword in tumor
development since complement activation is not only involved
in anti-tumor cytotoxicity and immune responses, but
also promotes tumor development directly and indirectly.
Regarding to its anti-tumor side, complement, upon
activation, displays various controlling effects (e.g. C3b/iC3b
mediated phagocytosis and TCC mediated cell lysis) on various
tumor cells including both solid tumors and hematological
malignancies (23). For example, upon treatment of CLL,
complement activation was initiated by anti-CD20
monoclonal antibody RTX, thereby displaying efficient CDC
and CDCP to clear the tumor cells (24). However, in order to
block the toxic effects of activated complement, and to survive,
tumor cells, similar to infectious microbes utilize multiple
evasion strategies to actively escape complement attack and
immune surveillance. For example, lung, ovarian, glial and
hematological tumor cells show enhanced expression and
surface binding of soluble regulators including Factor H,
FHL-1, FHR1, FHR-4, FHR5, and C4BP. These up-regulated/
bound complement regulators further display the cofactor
activity, which function together with factor I to block
complement activation at the level of C3 convertase, thereby
leading to complement evasion (25–29). Similarly, the
membrane-bound complement inhibitors (e.g. CD46, CD55,
and CD59) are up-regulated in various primary tumors and
FIGURE 1 | Complement activation, effector function and regulation. Complement system is activated by three different pathways, then merged at the level of C3
cleavage, followed by C5 convertase formation and generation of terminal complement complex. Upon activation, different activation products are generated, which
display multiple immune effector functions. The whole system is tightly self-controlled by different regulators.
Luo et al. Hematological Malignancies and Complement
Frontiers in Immunology | www.frontiersin.org October 2020 | Volume 11 | Article 5936103

tumor lines to evade the complement attack (30,31). Serglycin is
another endogenous complement inhibitor secreted by human
MM cell lines. Serglycin can inhibit both the classical and lectin
pathways’activation by direct interaction with C1q and
mannose-binding lectin, thereby blocking complement
mediated immune effector effects on MM cells (32).
Complement Mediated Promotion
of Tumor Growth
Besides the controlling effect, over-activation of complement also
promotes tumor growth through the pro-inflammatory
properties of effector compounds, which is in line with
established cancer-promoting effects of chronic infections (33).
Chou et al. reported that local production and activation of
complement effector compounds were distinctly important for
promoting tumor growth, while the systemic production of
complement by liver did not affect cancer (34).
C3a–C3aR and C5a–C5aR Mediated
Proliferative Effects
The proliferative effect on tumors is directly correlated to
the activation of complement cascade, as derived by C3a and
C5a via C3aR and C5aR1 mediated PI3K/AKT signaling
pathway (34). Shu et al. reported that C3a-C3aR signaling via
PI3K/AKT pathway in carcinoma associated fibroblasts
facilitated the metastasis of breast cancer. Targeting C3aR
signaling was shown to be a potential anti-metastasis
strategy for breast cancer therapy (35). Upon signaling by C5a
stimulation, C5aR1 expressed tumor cells underwent cytoskeletal
rearrangement (e.g. filopodia formation, membrane ruffling) and
furthermore released matrix metalloproteinase, which lead to
increased tumor cell motility and invasiveness both in vitro and
in vivo (36). Another report showed that C5aR1 signaling
induced myeloid-derived suppressor cells to produce larger
amounts of reactive oxygen species and reactive nitrogen
species, which inhibited CD8+ T cell mediated anti-tumor
activity, thereby leading to tumor growth (37). Vadrevu et al.
showed that in a lung cancer metastatic model, C5aR1 blockage
resulted in decreased lung metastasis due to reduced TGF-b and
IL-10 production by myeloid-derived suppressor cells since both
TGF-b and IL-10 induced regulatory T-cell generation and
facilitated an immunosuppressive Th2-based T cell responses
(38). Further in vivo analysis showed that C5aR1 signaling
promotes melanoma growth by promoting infiltration of
immunosuppressive leukocyte populations into the tumor
microenvironment, whereas C5aR2 has a more restricted but
beneficial role in limiting tumor growth, further proving the
“double-edged”role of complement activation in tumor
promotion (39).
Other Complement Components
Mediated Effects
Besides the complement receptors mediated effects, different
components of the main cascade also display onco-progression
ability, such as C1q, C3 and C5b-9. Bioinformatics analysis by
Mangogna et al. showed that high levels of C1q have a favorable
prognostic index in basal-like breast cancer for disease-free
survival, and in HER2-positive breast cancer for overall
survival (40). C1q acts in the tumor microenvironment as a
cancer-promoting factor in a complement dependent as well as
independent manner. In clear-cell renal cell carcinoma, tumor
associated macrophages produce high densities of C1q, which
together with tumor cell expressed C1r, C1s, C4, and C3 initiates
CP activation. The activation products as well as tumor
associated macrophages-derived C1q further promotes an
immunosuppressed microenvironment characterized by high
expression of immune checkpoints (e.g. programmed cell death
protein (PD)-1, Lag-3, programmed cell death ligand 1 (PD-L1),
and PD-L2), thereby fueling tumor progression. Mice deficient in
C1q, C3, and C4 displayed decreased tumor growth (41).
Yuan et al. reported that local complement C3 overexpression
activated JAK2/STAT3 pathway and promoted gastric cancer
progression. Further clinical investigation showed that C3 and
C3a expression was markedly enhanced in gastric cancer
tissues at both mRNA and protein levels compared with those
in paired non-tumorous tissues. High C3 deposition was
identified as an independent prognostic factor of poor 5-year
overall survival, suggesting that local C3 deposition in the tumor
microenvironment is a relevant immune signature for predicting
prognosis of gastric cancer (42). In vivo data showed that the
knockdown of C3 suppressed hepatic stellate cells-promoted
hepatocellular carcinoma development (43).
Further, sublytic C5b-9 displays tumor-promoting properties
by activating signal transduction pathways (e.g., Gi protein/
PI3K/Akt kinase and Ras/Raf1/ERK1) and modulating the
activation of cancer-related transcription factors, while shielding
malignant cells from apoptosis (44). Such complement promoted
tumor progression also actively involved in cutaneous squamous
cell carcinoma, which was nicely reviewed by Pilvi Riihilä
et al. (45).
Based on these roles of complement in promoting cancer
progression, more and more complement proteins are becoming
potential candidates for cancer targeted therapy and numerous
new anti-complement drugs are under clinical development (46).
For example, C3aR and C5aR are recently classified as a new class
of immune checkpoint receptor in cancer immunotherapy (47).
In addition, humanized soluble CR1-Fc fusion protein were
generated to target C3b/C4b and its therapeutic effect was
conferred in a colitis-associated colorectal cancer model and
orthotopic 4T1 breast cancer model (48).
COMPLEMENT IN HEMATOLOGICAL
MALIGNANCIES
The number of patients with hematological malignancies is
increasing on a daily basis. Many patients with hematologic
malignancies display abnormal levels of complement components,
possibly as a result of the tumor avoiding complement surveillance
(Figure 2).Inthispart,wewillsummarizetheroleofcomplement
in hematological malignancy conditions.
Luo et al. Hematological Malignancies and Complement
Frontiers in Immunology | www.frontiersin.org October 2020 | Volume 11 | Article 5936104

Complement in Multiple Myeloma
MM is a hematological malignant tumor characterized by clonal
proliferation of plasma cells that produce M protein,
accompanied by various types of impaired immune function.
C1q, being one of the main components in CP, seems to be a
potential marker of the immunodeficiency status in MM
patients. Statistically, the mean serum level of C1q in MM
patients is lower than that in the control group. At different
stages of MM, C1q level also changes dynamically. When the
disease is in remission, C1q is back to a normal level. However,
with disease re-progressed, C1q level in the plasma decrease
again, which indicates that MM cells, by controlling/consuming
C1q, to some extent modulate the CP activation (49). Further,
MM cells recruit complement regulator Factor H to their surface.
Surface bound Factor H mediates factor I to cleave C3b, thereby
down-regulating complement activation. Consistent with the
above findings regarding complement deficiency, evidence
shows that MM patients are at high risk of bacterial infections.
No matter with normal or elevated level of C3, all sera from MM
patients have a defect in C3b binding to Streptococcus
penumoniae. Experimental data showed that addition of
normal human serum to serum from MM patients restored the
C3b binding ability to S. pneumoniae. However, adding anti-S.
pneumoniae antibodies to MM serum did not rescue C3b
deposition to any S. pneumoniae types. Such a scenario
indicates that serum from MM patients has a defect in C3
activation, also explains why the MM patients have increased
susceptibility to S. pneumoniae infections (50). Further, a recent
clinical investigation showed that serumC3 and C4levels in MM
patients were significantly higher than those in healthy people.
Among the MM patients, serum C3 and C4 levels positively
correlated with diseases severity. However, the mechanism how
serum C3 and C4 promote MM development is not yet
clear (51).
Ficolins (Ficolin 1, 2, 3) are complement lectin pathway
defense factors, which are able to distinguish between
“self”,“abnormal self”and “non-self”, thereby contributing to
the elimination of “abnormal self”and “non-self’by direct
opsonization and/or initiation of complement activation
through the lectin pathway. Lower level of ficolin-1 and ficolin-2
as well as polymorphisms of FCN1 and FCN2 genes were detected
in patients diagnosed as MM, compared with control group,
which leads to the higher hospital infections because of limited
complement activation (52). The same group reported that in the
early time a higher frequency of mannose-binding lectin
deficiency-associated genotypes among MM patients was
detected compared with controls (53).
Complement in Leukemia
Leukemia is another type of life-threatening hematological
malignancy. Experimental data show that leukemia cell lines
together with clonogenic blasts from both CLL and acute
myeloid leukemia (AML) patients respond strongly to C3 and
C5 cleavage fragments in form of chemotaxis and increased
adhesion. C3aR and C5aR were also detected at the mRNA and
protein level in both human malignant hematopoietic cell lines
and patient blasts upon stimulation by C3a and C5a (54).
Further, abnormal C5 pattern approved in 42% CLL patients
since C5 protein was detected as double bands accompanied by a
lower molecular band when analyzed by Western blot, indicating
that C5 cleavage/C5 activation happened, whereas C5 from the
healthy controls and other CLL patients showed only one normal
FIGURE 2 | Complement evasion of hematological malignancies. Different types of hematological malignancies utilize different strategies for complement evasion.
In MM patients, serum levels of C1q are down-regulated, which mediates CP inactivation. MM cells recruit Factor H to their surface, thereby down-regulating AP
activation. Up-regulation of CD46 and CD55 inhibits complement activation. Down-regulation and variation of ficolin1, 2 lead to LP inactivation. In CLL patients,
tumor cells altered C5 pattern to modify CP activation. Variation of C9 correlates with EFS of FL, and variation of C7 correlates with EFS of DLBCL.
Luo et al. Hematological Malignancies and Complement
Frontiers in Immunology | www.frontiersin.org October 2020 | Volume 11 | Article 5936105

band (55). In comparison to patients with normal C5 pattern,
patients with abnormal C5 pattern have increased basal levels of
sC5b-9 and C5a, while sC5b-9 levels after CP activation were
significantly decreased by 48%, suggesting CP activation was
impaired. Such a scenario suggests a link between the pattern of
C5 and CP activation (55) in CLL patients. In addition, a defect
of C3b deposition on bacterial surface was detected although the
serum concentrations of C3 are normal in patients with CLL.
Mixing normal serum with serum from CLL patients restored
C3b binding to bacterial surface, suggesting a defect in either the
activation or activity of C3 in CLL serum, which likely accounts
for the increased incidence of infections in these patients (56).
Further, significant decrease of serum C1 and C4 levels was
found in CLL patients, which correlated with the abnormal
hemolytic activities. Meanwhile, a complement profile
characteristic of acquired C1-IHN deficiency was observed in
all tested patients, which indicates that the depression of the CP
activity is a frequently occurring feature in CLL patients (57).
Moreover, different patterns of complement proteins were
observed in different types of leukemia. In AML patients, each
complement parameter tested was elevated as compared to the
control values (sera of healthy blood donors). The extent of C3
activation and C3 splitting were correlated with disease severity
while immunoglobin level remained consistently high and not
varied much between different types of leukemia. Similar results
were observed in acute lymphocytic leukemia patients although
the differences were less marked. In chronic myelocytic
leukemia, the CP and AP activities behave differently:
activities of CP and serum C4 levels were significantly
elevated, whereas activity of AP as well as serum C3 and
factor B concentration were not significantly different from
the control groups (58).
CR1 is expressed by erythrocytes and most leukocytes. sCR1,
a soluble form, is shed from the leukocytes and found in plasma,
which is identified as a powerful inhibitor of complement. Sadallah
et al. reported sCR1 in the plasma of leukemia patients increased
up to levels producing measurable complement inhibition, which
is a possible complement evasion strategy utilized by leukemia
cells (59).
Complement in Non-Hodgkin Lymphoma
The most common types of NHL are DLBCL and FL. Germline
mutations in complement genes have been associated with
susceptibility to infections and autoimmune diseases,
conditions that are associated with NHL risk (60). For
example, genetic variations of complement genes (e.g., MASP2,
C5 and C9) are strongly associated with the development of NHL
(60,61). Charbonneau et al. also found that variations in C9 and
complement regulatory genes (e.g., Factor H, CFHR1, CFHR5,
CD46, and CD55) were associated with the event-free survival
(EFS) of FL, while C7 variations were associated with EFS of
DLBCL (62). Thus, patients with different genotypes of
complement components respond differently to complement
attack and to antibody-based immunotherapy. Factor H,
CFHR1 and CFHR5 polymorphisms have a stronger impact on
EFS of FL patients who were treated with anti-CD20 antibody,
while CD46 and CD55 polymorphisms had a stronger impact on
EFS in FL patients who did not receive initial treatment. In
addition, C1qA polymorphism seems to be a biomarker for
predicting first-line response to R-CHOP regimen in DLBCL
patients. In a retrospective analysis, DLBCL patients with C1qA
homozygote A allele showed higher complete remission rates
and longer overall survival after receiving R-CHOP regimen with
an unknown mechanism (63). Further, over-expression of
complement regulators, such as CD46, CD55 and CD59 is one
of the main strategies utilized by NHL cell lines for complement
evasion (64). All these data suggest a potential role of the
complement system in NHL progression.
The Co-Expression of Complement Genes
by Hematological Malignancies and Solid
Tumors
Many complement genes are co-expressed by different types
of tumors. Roumenina et al. reported that there is strong
heterogeneity in expression among different complement genes;
however, not much difference existed between cancer types when
comparing the transcription levels of 50 complement related genes
within 30 types of tumors (mainly solid tumors) (65). When we
further compared the co-expression profile of complement genes
between hematological malignancies and solid tumors using the
available data, both DLBCL and AML have higher expression level
of complement CFD gene. Moreover, similar to most of the solid
tumors, DLBCL also shows higher expression of C3,ITGB2,as
well as genes of the classical pathway (i.e. C1QA,C1QB,C1QC,
C1R,C1S,andC2), while the C4BP, lectin pathway genes (i.e.
MBL2, FCN,andMASP2), and the terminal pathway genes (i.e C6,
C8. and C9) are poorly expressed. However, different transcription
profiles of the highly expressed genes are observed for the AML.
Besides CFD,FCN1, C1RL, C3AR1, and C5AR1 are strongly
expressed, the rests including C3 are all poorly expressed
(Supplementary Figure 1,Supplementary Table 1). In
addition, Roumenina L. and colleagues suggested that the co-
expression of complement genes by the tumors confers poor or
favorable prognosis or had no impact depending on the cancer
type. In this scenario, both DLBC and AML fall in the group of “no
impact on the prognosis”based on our further analysis
(Supplementary Figure 2,Supplementary Table 2).
ANTIBODY-BASED IMMUNOTHERAPY
AGAINST HEMATOLOGICAL
MALIGNANCIES
Different Types of Immunotherapies for
the Treatment of Hematological
Malignancies
Immunotherapy is becoming the mainstream for many types of
hematological. malignancies. Hematological malignancies are
derived from immune cells and are “sitting”in immune
microenvironment, thereby having many opportunities to
interact with resident immune cells, local antibodies,
or complement system. This provides unique opportunities
Luo et al. Hematological Malignancies and Complement
Frontiers in Immunology | www.frontiersin.org October 2020 | Volume 11 | Article 5936106

for immunotherapy. Currently, immunotherapy against
hematological malignancies involves two major approaches: T
cell therapies and antibody therapies. For T cell therapies,
chimeric antigen receptor T (CAR-T) cell therapy is
particularly promising for hematologic malignancies, garnering
two FDA approvals using autologous cells in 2017 (66,67), one
for the treatment of pediatric ALL and the other for adult
patients with advanced lymphomas (68). The main point for
CAR-T therapy is generating engineered T cell by transfection of
mRNA for CAR domain expression which typically consists an
antigen-binding domain, a hinge that connects the scFv to a
transmembrane domain and a signaling domain composed of
CD3z. Antibody therapies include mAb and bispecific antibody.
Blockade of PD-L1/PD-1 interaction has brought about another
advance in immunotherapy for hematological malignancies. The
clinical outcomes of anti-PD-1 mAbs on Hodgkin’s lymphoma
are particularly impressive (69,70). Combining mAb and CAR-T
cell therapy, bispecific antibodies (BsAbs) were further
developed, which contains one tumor antigen binding side and
another side for binding and activating T cells. Due to the
structure specificity, BsAbs can bridge T cells and target cells,
thereby redirecting and activating T cell at sites of tumor cells
(71). Currently, FBTA05 (Lymphomun) is a heterodimeric BsAb
that recognizes CD20 and CD3, which has been used as
monotherapy or followed by donor lymphocyte infusion in the
treatment of CLL, high grade NHL, ALL, post-transplant
lympho- proliferative disease. In addition, CD123- and CD33-
specific BsAbs have been evaluated in clinical trials for patients
with AML (72).
Current Existing Monoclonal Antibodies
Against Hematological Malignancies
Over the past two decades, the use of mAb and molecules derived
from them has achieved considerable attention and success,
establishing this mode of therapy as important therapeutic
strategy in many cancers, especially in hematological
malignancies. Among mAbs used for the treatment of
hematological malignancies, anti-CD20 is the most routinely
used and well characterized mAb for the treatment of the CD20-
positive NHL and chronic CLL with major therapeutic
advances (73). RTX, ofatumumab, ublituximab, veltuzumab,
ocaratuzumab as well as tositumomab, obinutuzumab are
currently available types. In addition, 97% of patients with
classical HL typically exhibit an overexpression of PD-L1 due
to the alteration in chromosome 9p24.1 (74). Therefore, the PD-
1/PD-L1 axis is a good target for mAbs to kill tumor cells in HL.
Nivolumab, a human IgG4 mAb, blocks the interaction of PD-L1
and PD-L2 by binding to the PD-1 receptor on activated immune
cells, which was already approved by the FDA in 2016 for the
treatment of relapsed or progressed HL (75). The anti-CD33 (e.g.
immunotoxin, gemtuzumab, and ozogamicin) and anti-CD52
mAbs (alemtuzumab) are approved for treatment of relapsed
AML in older patients and B-cell CLL. IGN523, targeting CD90
on the surface of malignant hematological cells (e.g. AML) is
currently being evaluated in a Phase I clinical trial for AML (76).
In addition, monoclonal antibodies targeting CD4, CD19, CD20,
CD22, CD23, CD25, CD45, CD66, and CD122 are also under
investigation in the clinic for the treatment of leukemia (77).
Breakthrough has also been made in targeting surface
molecules expressed by MM cells, such as daratumumab,
isatuximab, MOR202 as well as SAR650984 (different
generations of humanized anti-CD38 monoclonal antibodies)
(78), and elotuzumab, a humanized anti-signaling lymphocytic
activation molecule family member 7 mAb. Among these mAbs,
daratumumab and elotuzumab have been approved in the
treatment of relapsed or refractory MM patients who received
at least three prior therapies including proteasome inhibitors and
immunomodulatory drugs (79). Investigational mAbs targeting
CD138, CD56, CD40, CD74, BAFF, BCMA, GRP78, IGF-1R,
and ICAM-1 on the surface of MM cells are pre-clinically
developed, and several of them are in clinical trials (80).
Effect of Complement on mAb-Based
Immunotherapies
CDC and CDCP display a vital role in mAb-based
immunotherapies (81). Approved for the treatment of
hematological malignancies, most mAbs make use of
complement in their mechanism of action. Upon application
of mAbs, the complement pathways need to be fully effective to
achieve better clinic efficacy. However, complement deficiencies,
the over-expression of membrane complement regulatory proteins
(e.g. CD55, CD59, and CD46) and fluid phase inhibitors (e.g.
CFH, CFHR5, and C4BP) in the tumor microenvironment often
cause resistance and non-responsiveness to mAb treatment (64,
82–84).
Di Gaetano et al. reported that the C1q-deficient mice
exhibited a defective response to RTX in a lymphoma tumor
mouse model. The work delineates the importance of CDC as an
important effector mechanism in immunotherapy instead of
ADCC as such an effect was not detected after depletion of
either natural killer cells or granulocyte cells (85). Further, CLL
patients undergoing complement deficiencies are suspected of
limiting anti-CD20 mAb efficacy in vivo (83). In CLL, upon
administration of RTX or ofatumumab, complement is quickly
consumed (86,87). C1 and C4 levels were below normal in more
than 50% of the sera tested from CLL patients (57). Only when
concurrent administration of fresh frozen plasma that RTX
therapeutic activity can be restored in CLL patients.
Polymorphisms of C1qA, C5, and C9 were often detected in
FL and DLBCL patients, which may also limit complement
activation, thereby affecting the clinical response to RTX (88).
Furthermore, complement regulators (CD55, CD59 and
Factor H) limited RTX efficacy in CLL patients via down-
regulating CDC (89). Functional block of these regulators
significantly increased the susceptibility of primary CLL cells to
anti-CD20 mAb. In FL and DLBCL patients, CD46, CD55,
CD59, CFH, CFHR1, and CFHR5 gene expression likely affects
the clinical response and duration of response to RTX therapy
(90). Like CLL and NHL, MM cells also utilize complement
regulators (CD55, CD59, and CFH) to block CDC, thereby
resisting anti-CD38 mediated-antitumor immunotherapy (91).
The serum levels of serglycin are elevated in patients with MM
Luo et al. Hematological Malignancies and Complement
Frontiers in Immunology | www.frontiersin.org October 2020 | Volume 11 | Article 5936107

compared to healthy controls. Upon mAb treatment, serglycin
protects MM cells from complement attack by blocking CDC,
thereby promoting survival of malignant cells (32). Due to the
different expression levels of each regulator/inhibitor of
individual patients, mAb efficacy varies from patient to patient.
All in all, these comprehensive investigations that were
performed in vitro studies and in mouse models as well as
analyses from clinical patients provide key insights that functional
complement is important for the mAb-based anti-tumor effects
on hematological malignancies (62,63). Down-regulation or
inhibition of complement activation by hematological tumors is a
big challenge for mAb-based immunotherapy.
Improvement of Clinical Efficiency of mAb-
Based Immunotherapies
To improve the clinical efficiency of mAbs, new strategies should
be developed to initiate efficient complement activation by (1)
mAb modifications (such as changing the target epitopes, Fc
mutation, and immunoglobulin G subclass switching), (2) the
control of membrane and soluble complement inhibitors, and (3)
the concurrent administration of fresh frozen plasma during
mAb therapy (84,92).
Despite the clinical success achieved with RTX, incomplete
treatment responses and emergence of resistance represent
important limitations, suggesting further improvements of
anti-CD20 mAb efficacy are required. Many novel anti-
CD20 antibodies are under development either by changing
the target CD20 epitope or by altering the Fc region to
enhance immune effector cell activity (ADCC, ADCP). For
example, compared with RTX, ofatumumab exhibits increased
CDC by binding to a different CD20 epitope (93), while by Fc
alterations, obinutuzumab has increased ADCC, reduced CDC,
and enhanced direct non-apoptotic cell death (94,95). CDC
induction by obinutuzumab is 10 to 100-fold less than by the
RTX and ofatumumab (93), resulting in a further-increased
capacity to bind and activate natural killer cells in the presence
of complement (96). In fact, Fc region engineering includes
modifying the amino acid sequence or the glycosylation
pattern, which allows enhancing both CDC and ADCC
effector functions. Using this technique, four variants of RTX
were generated as a native IgG1, a variant carrying the EFTAE
modification (S267E/H268F/S324T/G236A/I332E) for enhanced
CDC as well as glyco-engineered, non-fucosylated derivatives of
both to boost ADCC. Antibodies with EFTAE modification were
more efficacious in inducing CDC than antibodies with wild-type
sequences due to enhanced C1q binding. Meanwhile, non-
fucosylated variants had an enhanced affinity to FcgRIIIA and
improved ADCC activity (97).
So far several types of anti-CD20 mAbs have been developed,
such as RTX, ofatumumab (OFA), ublituximab, veltuzumab,
ocaratuzumab as well as tositumomab and obinutuzumab.
These variant anti-CD20 mAbs display different clinical
efficiency because of CD20-binding characteristics and ability
to induce CDC as well as ADCC (94). Structural analysis of
CD20 by RougeL. et al. nicely explains why some anti-CD20s are
more efficient complement activators than others. The authors
show that CD20 exists as a compact double-barrel dimer, which
can be bound by two RTX antigen-binding fragments (Fabs).
Each of the dimerized CD20 consists two parts, one as a
composite epitope and the other one as an extensive
homotypic Fab: Fab interface. RTX, by cross-linking CD20
into circular assemblies forms a structural model for
complement recruitment, thereby leading to stronger
complement activation (98).KumarA.andhiscolleagues
further used the cryo-electron microscopy to identify
structures of full-length CD20 complexed either with
prototypical type I (RTX and OFA) or type II (Obinutuzumab)
mAbs. Their data showed that type I complexes function as
molecular seeds to increase local concentration of mAbs, thereby
effectively activating complement upon binding to CD20, which
is similar to what RougeL. et al. reported. However, type
II complexes are unable to recruit additional mAbs and
complement components, thereby failing to cause efficient
complement activation. In addition, compared to RTX, OFA
activates complement more potently because of the sharper
binding angle of Fab
OFA
, suggesting that concatenation of
IgG
OFA
seeding complexes may bring their Fc domains in
closer proximity, further facilitating their oligomerization (99).
Human IgG3 activates complement most efficiently among
the IgG subclasses. An IgG3 switch variant of RTX induced
better CDC even on low CD20 expressing cells compared with its
parental IgG1 counterpart (100). Further, the shorter serum half-
life of IgG3 can be rescued by the introduction of the R435H
mutation, resulting in a potent mAb for CDC (101). IgG1/IgG3
chimera targeting CD20 showed stronger C1q binding, increased
CDC capacity and more efficient B cell depletion in cynomolgus
monkeys compared with the isotype matched parental mAbs
(102,103).
Furthermore, application of mAb together with inhibitors to
mCRP as an adjuvant can achieve higher efficiency. The ongoing
TABLE 1 | Complement-related therapies for treatment of hematological malignancies.
Therapies Functions Targeted molecules Molecular nature References
rILYd4 blocking CD59 regulatory function CD59 30 amino acid fragments (104,105)
hSCR18-20 blocking Factor H regulatory function Factor H recombinant protein (106,107)
MB-59 blocking CD59 regulatory function CD59 mini antibody (108)
MB-55 blocking CD55 regulatory function CD55 mini antibody (108)
mAb A247 blocking Factor I function Factor I neutrolize antibody (102,109)
Sorafenib decreasing the expression of complement regulatory Proteins unknown an oral compound (110)
ATRA decreasing the expression of complement regulatory proteins unknown Metabolic intermediates of vitamin A (91)
ATRA, all-trans retinoic acid.
Luo et al. Hematological Malignancies and Complement
Frontiers in Immunology | www.frontiersin.org October 2020 | Volume 11 | Article 5936108

clinical trials of complement-related therapies for treatment of
hematological malignancies are summarized in Table 1.
Recombinant ILYd4, a novel CD59 inhibitor, effectively
enhances the RTX-mediated CDC effect on RTX-sensitive RL-
7 lymphoma cells and RTX-induced resistant RR51.2 cells.
Meanwhile, recombinant ILYd4 also enhances the effect of
RTX and anti-CD24 mAb on the refractory MM cell line
ARH-77 (104,105). Similarly, sorafenib potentiates RTX and
ofatumumab efficacy in CLL and HL patients by decreasing the
expression of complement regulatory proteins. Such effect of
sorafenib has been investigated in more than 500 clinical trials
with promising activity and good patients’tolerance (110).
Alternatively, mCRP function could be blocked by specific
“neutralizing”antibodies. Blockage of CD55 and to a lesser
extent of CD59 with specific antibodies in vitro significantly
increased CDC of B lymphoma cells by RTX (111). However, the
use of intact anti-mCRP antibodies in vivo may lead to CDC on
healthy host cells. Mini-antibodies (MB-55, MB-59), composed
of single-chain variable fragments to CD55 and CD59 and the
human hinge-CH2–CH3 domains of IgG1, did not induce CDC
themselves, but increased RTX mediated CDC by twofold in
vitro (108). Their application in an in vivo model of human
CD20 positive B cell lymphoma in SCID mice markedly
increased survival upon RTX treatment (102).
Moreover, neutralization of sCRPs is another way to enhance
CDC. In combination with ofatumumab or RTX, human
recombinant Factor H-derived short-consensus repeat 18–20
(hSCR18–20) increased susceptibility of primary CLL cells to
CDC by abrogating Factor H function on the surface of CLL cells
(106,107). The decay of C3b to iC3b is strongly mediated by
Factor I, for which most of the described CRPs exhibit cofactor
function. Application of RTX or ofatumumab together with a
neutralizing mAb against Factor I (mAb A247) increased CDC
on CD20-expressing cell lines and primary CLL samples (102,
109). In addition, all-trans retinoic acid was reported to up-
regulate CD38 expression level and down-regulate CD55 and
CD59 level in daratumab-resistant MM cells, thereby enhancing
the CDC effect on MM cells (91).
A clinical investigation by Xu et al. showed that application of
fresh frozen plasma together with RTX is an effective measure to
regain CDC effect upon the treatment of fludarabine refractory
CLL patients. Twenty-two patients were treated with two units of
fresh frozen plasma followed with RTX as a single agent, repeated
every 1–2 weeks with a total of four courses of the combined fresh
frozen plasma and RTX treatment. Sixteen patients (72.7%)
responded to treatment, and seven (31.8%) achieved a complete
remission. Three (13.6%) of them had no evidence of minimal
residual disease after treatment (112). Such a clinical investigation
suggests that the concurrent administration of fresh frozen plasma
during mAb therapy is an optional measure to increase the mAb-
induced clinical efficacy.
CONCLUSIONS
This overview not only serves a fundamental understanding
of the roles of complement in tumor progression and in mAb-
based immunotherapy, but importantly, also highlights
potential therapeutic targets/measures to improve the clinical
efficacy of mAbs against hematological malignancies and
further extrapolates this knowledge to other tumor related
diseases. However, the mechanisms how complement affects
hematological malignancies’development and which strategy
increases mAbs’efficacy most clinically relevant are still elusive
and need further investigation.
AUTHOR CONTRIBUTIONS
SL designed the outline of the manuscript, wrote, and reviewed
the manuscript. MW wrote and reviewed the manuscript. HW
revised the manuscript. DH wrote and reviewed the manuscript.
YH and PZ discussed the topic and outlines of the manuscript
and reviewed the text. All authors contributed to the article and
approved the submitted version.
FUNDING
This work was supported by the National Natural Science
Foundations of China (No. 81601747, 82070136 to SL and No.
31770983, 81974249 to DH).
SUPPLEMENTARY MATERIAL
The Supplementary Material for this article can be found online
at: https://www.frontiersin.org/articles/10.3389/fimmu.2020.
593610/full#supplementary-material
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Conflict of Interest: The authors declare that the research was conducted in the
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