Cytokine xxx (2016) xxx–xxx

Contents lists available at ScienceDirect

Cytokine
journal homepage: www.journals.elsevier.com/cytokine

Molecular mechanisms of action of anti-TNF-a agents – Comparison

among therapeutic TNF-a antagonists
Hiroki Mitoma a,⇑, Takahiko Horiuchi b, Hiroshi Tsukamoto c, Naoyasu Ueda d
a
Department of Clinical Immunology and Rheumatology/Infectious Disease, Kyushu University Hospital, Fukuoka 812-8582, Japan
b
Department of Internal Medicine, Kyushu University Beppu Hospital, Beppu 874-0838, Japan
c
Department of Internal Medicine, Shin-Kokura Hospital, Kitakyushu 803-8505, Japan
d
Department of Internal Medicine, Miyazaki Prefectural Miyazaki Hospital, Miyazaki 880-8510, Japan

a r t i c l e i n f o a b s t r a c t

Article history: Tumor necrosis factor (TNF)-a is a potent pro-inflammatory and pathological cytokines in inflammatory
Received 2 May 2016 diseases such as rheumatoid arthritis and inflammatory bowel diseases. Anti-TNF-a therapy has been
Received in revised form 16 August 2016 established as an efficacious therapeutic strategy in these diseases. In clinical settings, three monoclonal
Accepted 16 August 2016
anti-TNF-a full IgG1 antibodies infliximab, adalimumab, and golimumab, PEGylated Fab’ fragment of
Available online xxxx
anti-TNF-a antibody certolizumab pegol, extracellular domain of TNF receptor 2/IgG1-Fc fusion protein
etanercept, are almost equally effective for rheumatoid arthritis. Although monoclonal full IgG1 antibod-
Keywords:
ies are able to induce clinical and endoscopic remission in inflammatory bowel diseases, certolizumab
Tumor necrosis factor-a
Anti-TNF-a therapy
pegol without Fc portion has been shown to be less effective for inflammatory bowel diseases compared
Inflammatory bowel disease to full IgG1 antibodies. In addition, there are no evidences that etanercept leads clinical remission in
Rheumatoid arthritis inflammatory bowel diseases. Besides the common effect of anti-TNF-a agents on neutralization of
soluble TNF-a, each anti-TNF-a agent has its own distinctive pharmacological properties which cause
the difference in clinical efficacies. Here we focus on the distinctions of action of anti-TNF-a agents espe-
cially in following points; (1) blocking ability against ligands, transmembrane TNF-a and lymphotoxin,
(2) effects toward transmembrane TNF-a-expressing cells, (3) effects toward Fcc receptor-expressing
cells, (4) degradation and distribution in inflamed tissue. Accumulating evidence will give us the idea
how to modify anti-TNF-a agents to enhance the clinical efficacy in inflammatory diseases.
Ó 2016 Elsevier Ltd. All rights reserved.

1. Introduction transgenic mice, tmTNF was shown to be sufficient to induce sys-

TNF-a is a major pro-inflammatory cytokine exerting pleiotro-
pic effects on various cell types by activating intracellular signaling
nuclear factor kappa B (NF-kB), mitogen-activated protein kinase
(MAPK), and caspases [1]. It plays a critical role for the pathogen-
esis of systemic inflammatory diseases such as rheumatoid arthri-
tis (RA), ankylosing spondylitis, psoriatic arthritis, inflammatory
bowel diseases (IBD), and Behçet’s disease [2]. Not only a mature
form soluble TNF-a (sTNF), but also its precursor form transmem-
brane TNF-a (tmTNF), is involved in various inflammatory
responses. tmTNF exerts its biological function in a cell-to-cell
contact manner, which is distinct from the feature of sTNF that
can act at the sites remote from TNF-a-producing cells [3,4]. In
⇑ Corresponding author at: Department of Clinical Immunology and Rheumatol-
ogy/Infectious Disease, Kyushu University Hospital, 3-1-1 Maidashi, Higashi-ku,
Fukuoka 812-8582, Japan.
E-mail address: mitoma@intmed1.med.kyushu-u.ac.jp (H. Mitoma).
temic arthritis accompanied with synovial hyperplasia and inflam-
mation [5,6]. tmTNF acts as a ligand by binding to TNF-a receptors
as well as functions as a receptor that transmits outside-to-inside
(reverse) signals into tmTNF-bearing cells (TNF-a-producing cells)
[7]. It is therefore considered that tmTNF plays a critical role in
local inflammation [8–13].
Anti-TNF-a agents have been successfully introduced into the
treatment of inflammatory diseases such as RA, juvenile idiopathic
arthritis, psoriatic arthritis, psoriasis, Crohn’s disease (CD), ulcera-
tive colitis (UC), ankylosing spondylitis, and Behçet’s disease
[14,15]. Infliximab, adalimumab and golimumab are full IgG1
monoclonal antibodies (mAbs) against human TNF-a. Infliximab
is a chimeric mouse/human anti-TNF-a monoclonal antibody
(mAb) composed of a murine variable region and a human IgG1
constant region. Adalimumab and golimumab are fully humanized
anti-TNF-a mAbs, which is indistinguishable from the normal
human IgG1. Etanercept is a fusion protein composed of the 2
extracellular portion of the human TNF receptor 2 (TNF-R2) (p75

http://dx.doi.org/10.1016/j.cyto.2016.08.014
1043-4666/Ó 2016 Elsevier Ltd. All rights reserved.

Please cite this article in press as: H. Mitoma et al., Molecular mechanisms of action of anti-TNF-a agents – Comparison among therapeutic TNF-a antag-
onists, Cytokine (2016), http://dx.doi.org/10.1016/j.cyto.2016.08.014
2 H. Mitoma et al. / Cytokine xxx (2016) xxx–xxx
TNF receptor) and the Fc portion (hinge, CH2 and CH3 domains) of
human IgG1. Certolizumab is a Fab’ fragment of an anti-TNF-a
mAb and lacks the Fc portion. The hinge region of certolizumab
is covalently linked to 2 cross-linked chains of a 20-kDa of poly-
ethylene glycol, which is named as certolizumab pegol (Fig. 1)
[1,16].
Despite all of these anti-TNF-a agents are effective in RA, clini-
cal efficacies on CD and UC, are not equal among these agents [17].
Specifically, anti-TNF-a full mAbs infliximab, adalimumab and
golimumab have a potential to induce clinical and endoscopic
remission in IBD, however soluble TNF-R2/IgG-Fc etanercept does
not. Even though head-to-head comparative studies have not been
performed, PEGylated anti-TNF Fab’ fragment certolizumab pegol
is considered to be less effective compared to full mAbs [18–20].
In addition, PEGylated dimeric extracellular human TNF receptor
1 (TNF-R1), onercept, was not efficacious against CD in a random-
ized, double-blind, placebo-controlled trial [21]. Moreover, inflix-
imab induces granulomatous infections like tuberculosis more
frequently than etanercept [22,23].
Binding and neutralizing activities against sTNF are the critical
and common mechanism of action of anti-TNF-a agents. On the
other hand, recent studies have demonstrated that these agents
have additional biological effects against tmTNF- [24–33] and Fc
receptor-expressing cells [34,35]. In addition, there are distinct
features of anti-TNF-a agents in pharmacological half-life, distribu-
tion and degradation [36–38]. It would be helpful for the further
understanding of different clinical efficacies of anti-TNF-a agents
on IBD to discuss the biological functions of them comparatively.
We would like to review the following issues: (1) biological activ-
ities of sTNF and tmTNF, (2) blocking abilities of anti-TNF-a agents
against ligands tmTNF and lymphotoxin, (3) action of anti-TNF-a
Fig. 1. Structures of therapeutic anti-TNF-a agents. Infliximab is a mouse/human
chimeric monoclonal anti-TNF-a IgG1 antibody. Adalimumab and golimumab are
fully humanized monoclonal anti-TNF-a IgG1 antibodies. Etanercept is a fusion
protein of the extracellular domain of human TNF-R2 and the Fc region of IgG1.
Certolizumab pegol is a PEGylated Fab’ fragment of humanized monoclonal anti-
TNF-a antibody.
Please cite this article in press as: H. Mitoma et al., Molecular mechanisms of action of anti-TNF-a agents – Comparison among therapeutic TNF-a antag-
onists, Cytokine (2016), http://dx.doi.org/10.1016/j.cyto.2016.08.014
agents toward tmTNF-expressing cells (TNF-a-producing cells),
(4) action of anti-TNF-a agents on Fcc receptors (FccR)-bearing
cells, (5) distribution and degradation of anti-TNF-a agents in
inflamed tissue. These knowledges would help us to treat refrac-
tory patients with inflammatory diseases better and to develop
more efficient therapeutic agents in the future.
2. Biological activities of soluble TNF and trasmembrane TNF
TNF-a is generated as a precursor form called tmTNF, which is
expressed as a type II polypeptide consisted of 233 amino acid
residues (26-kDa) on cell surface of activated macrophages and
lymphocytes as well as other cell types [39–45]. After cleavage
by such metalloproteinases as TNF-a-converting enzyme (TACE)
between residues alanine76-valine77, sTNF of 157 amino acid resi-
dues (17-kDa) is released from the cell surface and mediates its
biological activities through type 1 and type 2 TNF receptors
(TNF-R1 also known as TNFRSF1A, CD120a, and TNF-R2 also known
as TNFRSF1B, CD120b, respectively) [46–48]. sTNF is a homotrimer
of 17-kDa monomers and tmTNF also exists as a homotrimer of 26-
kDa monomers on the cell surface [49]. tmTNF also binds TNF-R1
and TNF-R2, but its biological activities are supposed to be medi-
ated mainly through TNF-R2 [50]. After releasing sTNF by TACE
cleavage, the residual cytoplasmic domain of tmTNF is additionally
cleaved by homologues of signal peptide peptidase (SPPL) and
migrates into the nucleus of tmTNF-bearing cells and mediates
production of pro-inflammatory cytokines [51–53].
TNF-R1 is constitutively expressed on almost all nucleated cells,
whereas TNF-R2 is generally inducible and is preferentially
expressed on endothelial cells and hematopoietic cells [1,54]. Both
TNF receptors form of preassembled homotrimers on the cell sur-
face and are capable of binding intracellular adaptor proteins that
lead to activation of intricate intracellular signaling processes and
mediate pleiotropic effects of TNF-a [55,56]. Receptor-mediated
effects of sTNF and tmTNF can lead to activation of NF-jB or to
apoptosis, depending on the metabolic state of the cell [1]. The sig-
naling pathways initiated by TNF-R2 that may be the preferential
receptor for tmTNF, are less characterized compared to TNF-R1.
However, TNF-R2 appears to have both shared and opposing effects
to TNF-R1 and may be actively involved in the pathogenesis of
inflammatory diseases [57].
tmTNF acts as a bipolar molecule that transmits signals not only
as a ligand, but also as a receptor in a cell-to-cell contact fashion.
tmTNF-a-bearing cells transmit signals to targets when cells
directly contact with TNF-R1- and/or TNF-R2-expressing cells.
The biological activities into tmTNF-bearing cells are transmitted
via TNF receptors through tmTNF, called as ‘‘outside-to-inside
signal” or ‘‘reverse signal” [4]. In contrast to well-characterized
functions of tmTNF as a ligand, the biological functions elicited
by outside-to-inside (reverse) signal have not completely been
clarified. However, it is supposed that outside-to-inside signaling
mediated by tmTNF contributes to the pleiotropy of this pro-
inflammatory cytokine and its fine tuning of immune responses
[7]. The biological activities of tmTNF as a receptor have been
demonstrated in T lymphocytes, monocytes/macrophages and nat-
ural killer (NK) cells in humans [8–13]. The elevation of intracellu-
lar calcium concentration in both human T cell lines and murine
macrophage cell lines was induced through tmTNF [58,59].
3. Binding and neutralization of anti-TNF-a agents to soluble
and transmembrane TNF-a
Infliximab binds both monomeric and trimeric forms of sTNF,
whereas etanercept binds only to the trimeric forms of sTNF [30].
Each infliximab molecule is capable of binding two TNF-a
 H. Mitoma et al. / Cytokine xxx (2016) xxx–xxx
molecules, and up to three infliximab molecules can bind each
TNF-a homotrimer. In contrast, etanercept is supposed to forms
one-to-one complex with a TNF-a homotrimer [30]. In fact, only
mAbs, but not etanercept, form large protein complexes in vitro
[60]. Although several groups examined binding activities of anti-
TNF-a agents to sTNF in different assay systems, all of infliximab,
adalimumab, etanercept and certolizumab pegol have almost sim-
ilar intrinsic binding properties to sTNF and nearly equal neutral-
ization abilities against TNF receptor signaling [25,28]. Shealy D
and colleagues reported that the affinity of golimumab to sTNF is
superior to those of infliximab and adalimumab. Golimumab is
conformationally more stable, and the inhibitory ability of goli-
mumab against TNF-a-induced cytotoxicity and activation of
human endothelial cells is better than those of infliximab and adal-
imumab [31].
Infliximab, adalimumab, golimumab, etanercept and cer-
tolizumab pegol bind to tmTNF on tmTNF-transfected cells with
similar affinities that were lower compared to sTNF [25,28,31,
61,62]. Other groups reported that etanercept does not bind tmTNF
or binds much weakly compared to anti-TNF-a mAbs [33,34]. As
with sTNF, up to three molecules of infliximab can bind one tmTNF,
one etanercept can bind one molecule of tmTNF, suggesting that
anti-TNF-a mAbs forms large molecular complexes with tmTNF on
the cell membrane [30]. Therefore, quantitative differences in
binding between etanercept and anti-TNF-a mAbs may come from
stoichiometric differences rather than distinctions of affinity. In
terms of inhibition of ligand activities of tmTNF, infliximab was sig-
nificantly more potent than etanercept at blocking tmTNF-mediated
E-selectin expression in human umbilical vein endothelial cell
(HUVEC) [30]. In a bioassay using human lung carcinoma cell line
A549, infliximab, adalimumab, and certolizumab pegol similarly
inhibited tmTNF-mediated cell death, however etanercept showed
about 2-fold less activity [28]. Taken together, effector functions of
tmTNF as a ligand are inhibited by any of anti-TNF agents, although
the activity of etanercept is weaker than the other agents.
4. Binding and neutralization of anti-TNF-a agents to
lymphotoxin-a
The homotrimer of lymphotoxin-a (LTa3) and heterotrimer of
two lymphotoxin-a and one lymphotoxin-b (LTa2b1) bind both
TNF-R1 and TNF-R2. Only etanercept, but not infliximab and
adalimumab, can bind and neutralize LTa3 and LTa2b1 [1,25,30].
One lymphotoxin-a also forms heterotrimers (LTa1b2) with two
lymphotoxin-b which is much more dominant than LTa2b1
in vivo. LTa1b2 binds lymphotoxin-b receptor (LTbR) but not TNF-
receptors, hence etanercept is not able to neutralize LTa1b2 [63].
LTa3 mediates proliferation and pro-inflammatory cytokine
secretion of RA synovial fibroblasts and activates the inflammatory
environment in human chondrocytes [64,65]. Therefore, the neu-
tralization of LTa3 via etanercept is beneficial in patients with RA
to suppress inflammation [66]. However, anti-lymphotoxin-a
mAb, pateclizumab, was not effective against RA patients with an
inadequate response to disease-modifying anti-rheumatic drugs,
indicating that blocking of lymphotoxin-a alone is not sufficient
for controlling activities of RA [67].
The role of LTa3 in the pathogenesis of IBD, has not been well
documented. LTa1b2 has been reported to regulate the develop-
ment of intestinal lymphoid organs, including Peyer’s patches
and mesenteric lymph nodes. The intestinal inflammation in
chronic dextran sodium sulfate (DSS)-induced colitis is suppressed
by inhibition of LTbR signaling. Conversely, the course of Citrobac-
ter rodentium-induced infectious colitis is more severe in mice
with impaired LTbR signaling [68]. LTbR signaling via LTa1b2 might
play an important role in the intestinal inflammation, which
Please cite this article in press as: H. Mitoma et al., Molecular mechanisms of action of anti-TNF-a agents – Comparison among therapeutic TNF-a antag-
onists, Cytokine (2016), http://dx.doi.org/10.1016/j.cyto.2016.08.014
3
cannot be regulated by anti-TNF agents. Further studies are
required to understand the benefits of lymphotoxin antagonists
in the treatment of IBD.
5. Functional properties of anti-TNF-a agents on
transmembrane TNF-a-expressing cells
Considering the importance of tmTNF in the pathogenesis of
inflammatory diseases, distinct effects of anti-TNF-a agents on
tmTNF may explain the difference in these clinical efficacies at
least in part. A number of groups reported head-to-head compar-
ison of functional properties of these anti-TNF-a agents on
tmTNF-expressing cells [25–28,30–33].
5.1. Complement-dependent cytotoxicity (CDC)
In the system using human Jurkat T lymphocytes [27,32], mur-
ine NS0 myeloma cells [28], murine Sp2/0 myeloma cells [25], or
Chinese hamster ovary (CHO) cells [61], CDC via anti-TNF-a agents
was analyzed. Infliximab, adalimumab and golimumab induced
CDC much more potently than etanercept [25,27,28,32,61]. On
the other hand, certolizumab pegol did not have any CDC activities
[28,32], which is caused by the absence of Fc portion of IgG1. From
the structural point of view, lack of the activation of the comple-
ment cascade by etanercept seems to be intelligible as well. Inflix-
imab, adalimumab, golimumab and etanercept commonly possess
the Fc portion of IgG1, whose CH2 domain activates the first com-
ponent of complement (C1) activation. Etanercept does carry this
CH2 domain of IgG1, but not the CH1 domain. A narrow region
of 23 amino acid residues within the CH1 domain serves as a plat-
form of complement C3 activation [69,70], which was later con-
firmed that three amino acid residues within the specific 23
amino acids are involved in the covalent attachment with C3
[71–73]. Hence it is thus difficult for etanercept to make mem-
brane attack complex of complement proteins (C5b-C9) for CDC
at least in vitro. When activated human peripheral blood mononu-
clear cells (PBMCs) were studied as target cells, none of infliximab,
adalimumab and etanercept induced CDC [25], which may be due
to the use of different cell types from the above mentioned
experiments.
5.2. Antibody-dependent cell-mediated cytotoxicity (ADCC)
Infliximab, adalimumab, golimumab and etanercept showed
similar ADCC activities by using tmTNF-transfected Jurkat T lym-
phocytes as target cells and human PBMCs or NK cells as effector
cells [32], while infliximab and adalimumab showed much more
potent ADCC activities than etanercept in NS0 cells [28] or in
CHO cells [61]. Certolizumab pegol lacking Fc portion did not show
any ADCC activity in any cell types as speculated [28]. The discrep-
ancy in etanercept-induced ADCC is not clear, however it may be
caused by the different experimental conditions, such as species
and cell types of target cells, and expression levels of tmTNF. Inflix-
imab, adalimumab, golimumab and etanercept carry the CH2 and
CH3 domain of human IgG1, whereas certolizumab pegol does
not. These domains of IgG1 are involved in the binding to Fc recep-
tors of effector cells [74], which leads to the lysis of target cells by
the secretion of granzyme B and perforin from effector cells.
5.3. Outside-to-inside signaling (reverse signaling) into tmTNF-
expressing cells
Infliximab and adalimumab, but not etanercept, induced apop-
tosis and cell cycle G0/G1 arrest in tmTNF-expressing Jurkat T lym-
phocytes that neither express TNF-R1 nor TNF-R2. This effect is
4 H. Mitoma et al. / Cytokine xxx (2016) xxx–xxx
mediated by reverse signaling through tmTNF, independently of
complement and NK cells [26]. Despite the similar binding abilities
of anti-TNF-a mAbs to tmTNF, golimumab induced a significantly
weaker apoptosis than infliximab and adalimumab [32]. The rea-
son for this discrepancy is not clear, however one possible reason
might be the difference of epitopes among these antibodies. tmTNF
forms trimer on the cell surface, antibody can bind two TNF tri-
mers, leading to the accumulation of tmTNF on the cell membrane.
Supportively, cross-linking of etanercept bound to the cell-surface
tmTNF with anti-human IgG antibody resulted in an increased
apoptosis, which indicates that the multimer formation of tmTNF
on the cell surface is essential for the initiation of the subsequent
intracellular signals [26]. c-Jun N-terminal protein kinase (JNK)
activation followed by up-regulation of p21WAF1/CIP1, Bax and Bak
as well as reactive oxygen species (ROS) accumulation are impor-
tant intracellular signaling events for the apoptosis and cell cycle
arrest. In addition, site-directed mutagenesis revealed that three
serine residues in the cytoplasmic domain of tmTNF are essential
for these biologic effects [26]. The amino acid sequence of the
intracellular domain of tmTNF is well conserved in different spe-
cies. Moreover, all the three serine residues were conserved among
different species, indicating that these residues are critical for
physiological functions of tmTNF [59].
6. Induction of apoptosis by anti-TNF-a antibodies in lamina
propria T lymphocytes of Crohn’s disease, in psoriatic CD8+ T
cells, and in effector T cells against Mycobacterium tuberculosis
ten Hove T and colleagues studied induction of apoptosis in vivo
in ten patients with refractory CD via the biopsy of colon just
before and 24 h after the infusion of infliximab. A significant
increase in CD3 and TUNEL positive cells within the colonic biopsy
specimen was detected 24 h after infusion of infliximab. In this
study, detectable change of phenotype or apoptosis in peripheral
blood T lymphocyte was not observed [75]. They also tested apop-
tosis induction at 24 h after infusion of infliximab by administra-
tion of technetium labeled annexin V into infliximab-treated
patients, following visualization of apoptotic cells in vivo using a
single-photon emission computer tomography. Annexin V uptake
was specifically enhanced in the diseased intestine 24 h after infu-
sion of infliximab. Analysis of the mucosal biopsy specimens iden-
tified lamina propria CD4+ T cells as target cells of annexin V
undergoing apoptosis [76]. These facts suggest that infliximab
induced apoptosis of activated T lymphocytes in inflamed tissue.
Bedini C and colleagues investigated the apoptosis induction in
psoriatic skin-homing T-lymphocytes. Psoriatic CD8+ T lympho-
cytes, but not CD4+ T lymphocytes, were susceptible to
infliximab-induced apoptosis in vitro [77]. Caprioli F and colleagues
also investigated apoptosis in lamina propria in IBD patients
treated with infliximab. Infliximab-induced downregulation of
macrophages was significantly associated with both endoscopic
responses to the therapy and achievement of mucosal healing.
The reduction of lamina propria CD68+ macrophages was associ-
ated with an increased rate of macrophage apoptosis [78]. In the
clinical settings, it is likely that infliximab induces apoptosis at
least in part by tmTNF-mediated effects; CDC, ADCC and/or
outside-to-inside signaling even though several groups reported
that peripheral PBMCs from healthy donors did not show the
induction of apoptosis in vitro.
Infliximab was associated with twofold to eightfold greater
risks of granulomatous infections such as tuberculosis, listeriosis
and histoplasmosis than etanercept in post-marketing surveillance
in the USA [79]. The increased incidence of tuberculosis in patients
treated with infliximab or adalimumab compared to etanercept
was also reported in other European countries [22]. tmTNF has
Please cite this article in press as: H. Mitoma et al., Molecular mechanisms of action of anti-TNF-a agents – Comparison among therapeutic TNF-a antag-
onists, Cytokine (2016), http://dx.doi.org/10.1016/j.cyto.2016.08.014
been shown to contribute to the host defense against acute
Mycobacterium tuberculosis infection in humans. CD8+CCR7-
CD45RA+ effector memory T lymphocytes express granulysin and
mediate an antimicrobial activity against Mycobacterium tuberculo-
sis. This subset of T lymphocytes expressed tmTNF and bound
infliximab. Infliximab led them to be susceptible to complement-
mediated lysis and the resultant reduced an antimicrobial activity
[80].
Atreya R and colleagues raised another possibility for the induc-
tion of apoptosis in activated T lymphocytes. Infliximab, adali-
mumab and certolizumab pegol, but not etanercept, were able to
induce apoptosis of T lymphocytes in IBD only when mucosal
CD4+ T lymphocytes expressing TNF-R2 were co-cultured with
tmTNF-expressing CD14+ intestinal macrophages. The induction
of apoptosis was dependent on inhibition of TNF-R2 signaling in
CD4+ T lymphocytes. They proposed that CD14+ macrophages
protected CD4+ T lymphocytes from apoptosis through TNF-R2
survival signaling via tmTNF [81].
Even though identification of precise mechanisms of apoptosis
induction is extremely difficult, it has been established that
activated T lymphocytes or macrophages involved in pathogenesis
or host defense fall into apoptosis in vivo after infusion of anti-TNF-
a mAbs.
7. Expansion of regulatory T lymphocytes by anti-TNF-a
antibodies through enhancement TNF receptor 2 signaling
The role of TNF on the expansion and suppressive function of
regulatory T lymphocytes (Tregs) is still controversial in human
because a large number of contradictory findings have been
reported [82–90]. On the other hand, several studies have shown
that signaling through TNF-R2 is important for function of Tregs
in murine systems [91–94]. Nguyen DX and colleagues examined
effects of anti-TNF-a agents on human Tregs in RA and health con-
trols [95]. Monocytes from patients with RA expressed more
tmTNF on their surface than those from healthy controls. Adali-
mumab, but not etanercept, bound tmTNF on monocytes from RA
and further enhanced expression levels of tmTNF. This effect was
not observed in monocytes from healthy controls. Surprisingly,
adalimumab promoted the interaction between tmTNF on mono-
cytes and TNF-R2 on Tregs in RA, resulting in the expansion of
functional Foxp3-positive Tregs, which suppress Th17 lympho-
cytes. Etanercept did not expand Tregs at all in this RA system.
Infliximab, golimumab, and certolizumab pegol were not included
in this study. Precise mechanisms how adalimumab paradoxically
promoted TNF-R2 signaling mediated by tmTNF were not analyzed
in this study, thus further studies are required to understand the
significance of this effect in inflammatory diseases.
8. Functional properties of anti-TNF-a agents to
monocytes/macrophages through Fcc receptors
All of anti-TNF-a agents except certolizumab pegol contain Fc
portion of IgG1, hence these agents were speculated to exert sig-
nals through Fc receptors. During mixed lymphocyte reaction
(MLR), infliximab and adalimumab, but not etanercept and cer-
tolizumab pegol, reduced proliferation of T lymphocytes. This
effect was lost by blocking of Fc receptor using control IgG or by
deletion of Fc portion of anti-TNF-a mAbs. In addition,
certolizumab-IgG with Fc portion did inhibit T cell proliferation,
indicating that Fc portion is required for this inhibitory function.
In MLR, anti-TNF-a mAbs induced generation of a new population
of macrophages in an Fc-dependent manner. These macrophages
expressed the regulatory macrophage marker CD206, and had an
immunosuppressive phenotype, producing anti-inflammatory
 H. Mitoma et al. / Cytokine xxx (2016) xxx–xxx
cytokine interleukin (IL)-10 [34]. Importantly, they also showed
that the induction of regulatory macrophages was obtained in CD
patients with mucosal healing after treatment with infliximab,
but not in CD patients without mucosal healing. Authors discussed
that the different effect between infliximab and etanercept was
caused by lack of binding ability of etanercept to tmTNF on acti-
vated T lymphocytes [96]. It is still not clear why sTNF/etanercept
complexes is not enough to induce regulatory macrophages
through Fc receptor despite the presence of IgG1-Fc region. Arora
T and colleagues reported that upon addition of sTNF, anti-TNF-a
mAbs, but not etanercept, showed significant increased binding
ability to Fc receptors due to the formation of large protein com-
plexes by mAbs [61]. Thus, increased binding ability of anti-TNF-
a mAbs to Fc receptors mediated by sTNF/tmTNF recognition
might be an important factor for the induction of regulatory
macrophages.
On the other hand, Wojtal KA and colleagues reported unfavor-
able effects of anti-TNF-a mAbs through FccRI (CD64) in IBD
patients. Infliximab/TNF complexes activate Fc receptors on PBMCs
through tyrosine phosphorylation and induce the expression of
pro-inflammatory cytokines such as MCP-1, GM-CSF, IL-6 and IL-
8. Adalimumab showed the similar response, but certolizumab
pegol or F(ab’)2 fragments of infliximab did not have this activity,
confirming that Fc portion is required for this function. Etanercept
was not included in this study, hence we are not able to compare
this activity between antibodies and a soluble receptor. Colonic
mRNA expression levels of CD64 were significantly increased in
the inflamed tissues from non-responders of infliximab, compared
to responders of infliximab, indicating that CD64 signaling might
be one of mechanisms of non-responsiveness of anti-TNF-a mAbs
in CD [35].
Monocytes/macrophages express several kinds of activating Fc
receptors FccRI (CD64), FccRIIA (CD32), FccRIIIA (CD16) and one
inhibitory receptor FccRIIB (CD32) [97]. IgG1 can bind all of
these receptors, hence transmitting signaling in monocytes/
macrophages is complicated. The expression level of each receptor
will affect the output of Fc receptor signaling, therefore anti-TNF-a
mAbs exert an anti-inflammatory effect in some conditions and do
a pro-inflammatory effect in other conditions. FccR polymor-
phisms have been associated with clinical response to TNF-a
antagonists in patients with IBD [98–101], therefore, FcR signaling
might be one of important mechanisms of action of anti-TNF-a full
mAbs.
9. Serum half-life and binding ability of anti-TNF-a agents to
neonatal Fc receptors
Etanercept shows a shorter serum half-life (3–5.5 days) than
mAbs of infliximab (8–10 days), adalimumab (10–20 days), goli-
mumab (9–15 days) and certolizumab pegol (14 days) in vivo [1].
Neonatal Fc receptors (FcRn) bind to the Fc domain of IgG at acidic
pH in the endosome and protect IgG from the ongoing catabolic
activities, thereby contributing to the long serum half-life of IgG
[36]. The binding affinity of anti-TNF-a mAbs infliximab and adal-
imumab to FcRn was 10-fold higher than that of etanercept, result-
ing in the difference of serum half-life among anti-TNF-a agents
in vivo. Authors speculate that the glycosylation at the C-
terminus of TNF-R2 region might induce the Fc conformational
change or interference with the binding between Fc portion and
FcRn by the steric hindrance, resulting in the reduction of bindig
affinity of etanercept to FcRn [102]. Supportively, the affinity of
etanercept and mAbs became comparable after papain digestion
at hinge region, suggesting that differences in amino acid
sequences or posttranslational modification of the Fc portion did
not contribute to the difference in the binding affinity of these
Please cite this article in press as: H. Mitoma et al., Molecular mechanisms of action of anti-TNF-a agents – Comparison among therapeutic TNF-a antag-
onists, Cytokine (2016), http://dx.doi.org/10.1016/j.cyto.2016.08.014
5
agents to FcRn [36]. Certolizumab pegol lacking Fc portion was
confirmed not to have the binding ability to FcRn at all [103]. Goli-
mumab was reported to have the binding ability to FcRn, however
the comparative study with the other anti-TNF-a agents was not
performed [31].
Effects of binding with sTNF on the affinities of infliximab, adal-
imumab and etanercept to FcRn were examined by the surface
plasmon resonance sensorgrams. The affinity of infliximab–sTNF
complex or adalimumab–sTNF complex to FcRn was lower than
that of infliximab alone or adalimumab alone, respectively [36].
Interestingly, these results are opposite to the bindig affinity of
anti-TNF-a mAbs to Fcc receptors [61]. The affinity of etaner-
cept–sTNF complex to FcRn was comparable with that of etaner-
cept alone. These results suggest that for anti–TNF-a mAbs,
binding with target molecules decreases the affinity to FcRn.
Therefore, the anti–TNF-a mAbs complexed with TNF-a will be
degraded more rapidly than anti–TNF-a mAbs free from TNF-a
in vivo.
10. Proteolytic cleavage of anti-TNF-a agents by tissue
metalloproteinases
All of infliximab, adalimumab, golimumab, certolizumab pegol
and etanercept include the hinge region of human IgG1, which is
recognized by proteases such as matrix metalloproteinase
(MMP)-3 and MMP-12. Biancheri P and colleagues reported that
these metalloproteinases cleave infliximab and adalimumab into
Fc domain and Fab domain in vitro. Etanercept is cleaved into Fc
domain and extracellular domain of TNF-R2 by these proteases.
Certolizumab pegol and golimumab are not examined in this
study. Free F(ab’)2 fragments of infliximab and adalimumab after
proteolytic cleavage by MMP-3 or MMP-12 still can antagonize
functions of sTNF. On the other hand, soluble TNF-R2 isolated from
etanercept by these proteases lost the activity of neutralization of
sTNF. The precise mechanism of decreased neutralization ability
of etanercept after cleavage has been unknown. Dimeric form of
extracellular domain of TNF-R2 in etanercept might be broken
after cleavage, resulting in generation of monomer of TNF-R2.
Inflamed intestinal mucosal homogenates from patients with CD,
but not ones from health controls, cleaved these three anti-TNF-
a agents at hinge region. The authors also reported that the
presence of anti-hinge autoantibody in sera from patients
with CD. Cleaved hinge region become neo-antigen, leading to
the generation of autoantibodies against human IgG1. Sera from
non-responders to infliximab showed higher titer of anti-hinge
antibodies than one from responders to infliximab [37]. Although
it was not examined whether these anti-hinge autoantibodies
decrease neutralization abilities of anti-TNF-a agents against
TNF-a, proteolytic cleavage of anti-TNF-a agents by mucosal
metalloproteinases and anti-hinge autoantibodies might be one
of mechanisms which contribute to the unresponsiveness to anti-
TNF-a agents in IBD.
11. Distribution of anti-TNF-a agents into the inflamed tissue
Delivery of drugs at the site of inflammation is another impor-
tant factor for the efficacy of treatment in inflammatory diseases.
Palframan R and colleagues studied the distribution of infliximab,
adalimumab and certolizumab pegol labeled with the low molecu-
lar weight dye to the inflamed joints of mice with collagen-induced
arthritis using a biofluorescence imaging. Etanercept was not
included in this study. All of anti-TNF-a mAbs studied distributed
more effectively into inflamed joints than non-inflamed joints. The
ratio of distribution of certolizumab pegol into the inflamed joint
compared with the normal joint was greater than that observed
6 H. Mitoma et al. / Cytokine xxx (2016) xxx–xxx

Table 1
Pleiotropic mechanisms action of anti-TNF-a agents. Very strong, +++; strong, ++; weak, +; no effect; –; not determined, n.d.; soluble TNF-a; sTNF; tmTNF; transmembrane TNF-a;
lymphotoxin-a3, LTa3; complement-dependent cytotoxicity, CDC; antibody-dependent cell-mediated cytotoxicity, ADCC; regulatory T lymphocytes, Tregs; Fcc receptors, FccR;
neonatal Fc receptors, FcRn; matrix metalloproteinases, MMPs.

Actions of anti-TNF-a agents Full lgG1 mAb PEGylated TNF Reference

Fab’ receptor 2
Infliximab Adalimumab Golimumab Certolizumab Etanercept
(a) Binding and neutralization of
(1) sTNF ++ ++ +++ ++ ++ [25,28,30,31]
(2) tmTNF ++ ++ ++ ++ + [25,28,30,31,33,34,61,62]
(3) LTa3 – – – – ++ [30]
(b) Action toward tmTNF-expressing cells
(1) CDC ++ ++ ++ – + [25,27,28,32,61]
(2) ADCC ++ ++ ++ – +/++ [27,28,32,61]
(3) Apoptosis via reverse signaling ++ ++ + – – [26,32]
(c) Expansion of Tregs n.d. ++ n.d. n.d. – [95]
(d) Action toward FccR-bearing cells
(1) Induction of regulatory macrophages ++ ++ n.d. – – [34,96]
(2) Induction of pro-inflammatory cytokines ++ + n.d. – n.d. [35]
(e) Escape from catabolism through FcRn ++ ++ ++ – + [31,36,102,103]
(f) Cleavage by MMPs/loss of neutralization activity against sTNF +/ +/ n.d. n.d. +/+ [37]
(g) Distribution into the inflamed tissue + + n.d. ++ n.d. [38]

with infliximab and adalimumab. In addition, the duration of expo-
sure in the inflamed versus normal tissue was more prolonged for
certolizumab pegol than for both infliximab and adalimumab [38].
The further studies including all anti-TNF-a agents are expected in
experimental mice models of IBD such as the DSS-induced colitis
and the 2,4,6-trinitrobenzene sulfonic acid (TNBS)-induced colitis.
12. Conclusions
We have reviewed the diversity of functions of anti-TNF-a
agents introduced to the treatment of inflammatory diseases
(Table 1). Mechanisms of action of biological preparations are not
solely neutralization of target proteins, but also mediate additional
signals through unexpected receptors such as tmTNF and/or Fc
receptors. Biological functions of cytokines are complicated, hence
it is possible that blocking of cytokines induces bipolar effects
in vivo. Moreover, pharmacological properties of anti-TNF-a agents
such as the distribution to diseased tissues and the degradation by
proteases also contribute to the effects of biological preparations.
Understanding of mechanisms of action of anti-TNF-a agents and
the correlation with clinical aspects might enable us to select the
appropriate biological agents in individual cases and to develop
new biological preparations for inflammatory diseases.
Conflict of interest
The authors declare no conflict of interest.
Acknowledgements
This work was supported by in part by Grants-in-Aid for Scien-
tific Research from the Ministry of Education, Culture, Sports,
Science and Technology of Japan (26893188).
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