JOURNAL OF INTERFERON & CYTOKINE RESEARCH
Volume 35, Number 4, 2015
ª Mary Ann Liebert, Inc.
DOI: 10.1089/jir.2014.0125
Anti-CD138-Targeted Interferon Is a Potent Therapeutic
Against Multiple Myeloma
Esther M. Yoo,1 Kham R. Trinh,1 Danh Tran,1 Alex Vasuthasawat,1 Juan Zhang,1,2
Bao Hoang,3–5 Alan Lichtenstein,3–5 and Sherie L. Morrison1,6
Multiple myeloma (MM), a plasma cell malignancy, is the second most prevalent hematologic malignancy in
the US. Although much effort has been made trying to understand the etiology and the complexities of this
disease with the hope of developing effective therapies, MM remains incurable at this time. Because of their
antiproliferative and proapoptotic activities, interferons (IFNs) have been used to treat various malignancies,
including MM. Although some success has been observed, the inherent toxicities of IFNs limit their efficacy. To
address this problem, we produced anti-CD138 antibody fusion proteins containing either IFNa2 or a mutant
IFNa2 (IFNa2YNS) with the goal of targeting IFN to CD138-expressing cells, thereby achieving effective IFN
concentrations at the site of the tumor in the absence of toxicity. The fusion proteins inhibited the proliferation
and induced apoptosis of U266, ANBL-6, NCI-H929, and MM1-144 MM cell lines. The fusion proteins
decreased the expression of IFN regulatory factor 4 (IRF4) in U266. In addition, the fusion proteins were
effective against primary cells from MM patients, and treatment with fusion proteins prolonged survival in the
U266 murine model of MM. These studies show that IFNa antibody fusion proteins can be effective novel
therapeutics for the treatment of MM.
Introduction
M
ultiple myeloma (MM) is a disease characterized
by an excess of malignant plasma cells in the bone
marrow. Accumulation and proliferation of malignant myeloma cells result in the disruption of normal hematopoiesis
and changes to bone marrow vascularization and bone
physiology. Analyses of patient myeloma cells and human
myeloma cell lines (HMCLs) have revealed the extensive
molecular heterogeneity of this disease (Drexler and Matsuo
2000; Carrasco and others 2006; Lombardi and others 2006;
Moreaux and others 2011). The survival rate for MM is 7–8
years when patients are treated with drugs such as the proteasome inhibitor bortezomib, thalidomide, or lenalidomide,
which target myeloma cells in the bone marrow microenvironment (Anderson 2012). Currently there is no cure for MM.
Besides their antiviral and immunostimulatory activities,
interferons (IFNs) have antiproliferative activity and can
induce apoptosis in hematological malignancies and solid
tumors (Borden and others 2000; Borden and others 2007).
Many studies have shown that type I IFNs, which were the
first recombinant proteins used in the treatment of cancer,
may be highly effective against a variety of tumor cell targets (Borden and others 2007). However, the effectiveness
of type I IFNs for cancer therapy has been limited by their
short half-life of only 1 h (Peleg-Shulman and others 2004)
and associated side effects when used at high doses (Weiss
1998).
Previously, we have shown that antibody-IFN fusion
proteins are an effective strategy for targeting IFNs to cancer cells. Anti-CD20-IFN fusion proteins targeting lymphoma cells showed potent growth inhibitory activity both
in vitro and in vivo in murine lymphoma models (Xuan and
others 2010; Trinh and others 2013). In addition, fusion of
IFNa or IFNb to IgG increased its half-life to 8 h (Huang
and others 2007; Trinh and others 2013). Therefore, we
wanted to extend these studies to determine if targeted IFNa
would be effective against MM. In this initial study, we used
several different HMCLs, but focused our attention on the
U266 myeloma cell line. We constructed fusions of antiCD138 with IFNa2 and IFNa2YNS, a high affinity IFNa2
mutant. We chose CD138, also known as syndecan-1, as the
1
Department of Microbiology, Immunology and Molecular Genetics, University of California Los Angeles, Los Angeles, California.
State Key Laboratory of Natural Medicines, School of Life Science and Technology, China Pharmaceutical University, Nanjing, China.
3
Greater Los Angeles Veterans Administration Healthcare Center, Los Angeles, California.
4
Jonsson Comprehensive Cancer Center, Los Angeles, California.
5
Division of Hematology Oncology, University of California Los Angeles, Los Angeles, California.
6
Molecular Biology Institute, University of California Los Angeles, Los Angeles, California.
2
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target antigen. CD138 is a heparan sulfate proteoglycan that
is highly expressed on HMCLs and malignant plasma cells
in peripheral blood and in the bone marrow in patients
(Ridley and others 1993; Wijdenes and others 1996; Chilosi
and others 1999). Treatment with IFNa2 fusion proteins
resulted in decreases in cell viability. The mode of action of
the fusion proteins included the induction of apoptosis and
in U266 a decrease in expression of IFN regulatory factor 4
(IRF4), a protein which is required for MM cell survival. In
addition, the fusion proteins were effective against primary
patient cells and in vivo against U266 tumors in a murine
model. In some experiments the higher affinity anti-CD138IFNa2YNS protein showed increased antiproliferative activity over anti-CD138-IFNa2.
Materials and Methods
Cells
HMCLs cells were the generous gift of Dr. W. Michael
Kuehl and Dr. Diane Jelinek. Primary cells were obtained
after informed consent and approved by the institutional
medical ethics committee. Cells were cultured in the RPMI
1640 (Invitrogen, Carlsbad, CA) supplemented with 5%
fetal calf serum (FCS; Atlanta Biologics, Lawrenceville,
GA). Growth medium for ANBL-6 cells was supplemented
with 2 ng/mL of IL-6. Chinese Hamster Ovary (CHO) cells
were cultured in Iscove’s Modified Dulbecco’s Medium
(IMDM; Invitrogen) supplemented with 5% FCS.
Construction of expression vectors, protein
production, and purification
The heavy (H) and light (L) chain variable (V) region
amino acid sequences of the anti-CD138 antibody B-B4 were
obtained from US Patent Application No: 2009/0175863 and
used to construct IgG1, k proteins.
VH sequence: MGWSYIILFLVATATGVHSQVQLQQ
SGSELMMPGASVKISCKATGYTFSNYWIQRPGHGLEW
IGEILPGTGRTIYNEKFKGKATFTADISSNTVQMQLSSL
TSEDSAVYYCARRDYYGNFYYAMDYWGQGTSVTVSS.
VL sequence: MKSQTQVFIFLLLCVSGAHGDIQMTQ
STSSLSASLGDRVTISCSASQGINNYLNWYQQKPDGT
VELLIYYTSTLQSGVPSRFSGSGSGTDYSLTISNLEPE
DIGTYYCQQYSKLPRTFGGGTKLEIK.
The DNA sequence encoding a signal peptide was added
5¢ of the H chain and L chain V regions (MGWSYIILFLVATATGVHS and MKSQTQVFIFLLLCVSGAHG, respectively) as well as the nucleotide sequence containing a
Kozak ribosomal recognition site (5¢-GGATATCCACC-3¢).
To facilitate downstream cloning, the sequence 5¢-GCTA
GCC-3¢ was added 3¢ of the H chain V region, and the
sequence 5¢-CGTAAGTCGACG-3¢ was added 3¢ of the L
chain V region. The DNA sequence was synthesized using
codons optimized for CHO expression (DNA2.0, Menlo
Park, CA).
The L chain V region flanked by EcoRV and SalI restriction sites was sequence verified before cloning into an
expression vector containing the human k L chain constant
region. The H chain V region flanked by EcoRV and NheI
restriction sites was sequence verified and cloned into an
expression vector containing the human g1 H chain constant
region. Expression of the H and L chains yielded antiCD138 IgG1. To produce the fusion protein, the anti-CD138
YOO ET AL.
VH was cloned into an expression vector containing the
human g1 H chain, a Gly-Ser linker (SGGGGS), followed
by human IFNa2. This expression vector was named
pAH6905.
To construct the DNA vector for the expression of antiCD138-IFNa2YNS, nested PCR was used to introduce 3 amino
acid mutations—H57Y, E58N, and Q61S. The first round
of PCR was done using the forward primer 5¢-CGCGGA
TCCTGTGATCTGCCTCAAACCCAC-3¢ and reverse primer 5¢-CCTCTAGAATCATTCCTTACTTCTTAAACT-3¢.
The nested PCR was done using forward primer 5¢-CTCTA
CAATATGATCTCACAGATC-3¢ and reverse primer 5¢GATCTGTGAGATCATATTGTAGAG-3¢, which contains
the mutations to IFNa2. The insert was cloned into pCR2.1TOPO vector (Invitrogen) and the DNA sequence was
verified. The XbaI/BamHI fragment containing the mutant
IFNa2YNS sequence was cloned into an intermediate
human g1 H chain vector and named pAH11015. The BamHI/
AvrII fragment from pAH11015 containing the mutations to
IFNa2 was then used to replace the wild-type IFNa2 sequence
from expression vector pAH6905 (see previous paragraph).
Protein production and purification
Anti-CD138 IgG and fusion proteins were produced in
CHO cells by transfection of H and L chain expression
vectors. Stably transfected cells were selected with 1 mM
histidinol. To produce IgG and the fusion proteins, CHO
transfectants were seeded into roller bottles. At confluency,
cells were expanded to 100 mL with IMDM + 1% Fetal
Clone (Thermo Fisher Scientific, Waltham, MA). The supernatant was removed every 2–3 days and replaced with
fresh medium. Cell-free culture supernatants were then
passed through a protein A-Sepharose 4B fast flow column
(Sigma-Aldrich, St. Louis, MO) and the bound protein
eluted with 0.1 M citric acid, pH 3.5. Eluted fractions were
neutralized immediately with 2 M Tris-HCl pH 8.0.
Fractions were run on SDS-PAGE gels and stained with
Coomassie blue to verify protein purity and integrity.
Protein concentrations were determined using the BCA
assay (Pierce, Rockford, IL). Anti-CD20-IFNa2, anti-CD20IFNa2YNS, and anti-dansyl (DNS)-IFNa2 used as untargeted
control proteins were produced as described previously
(Xuan and others 2010).
MTS assay to determine metabolic activity
HMCLs were seeded in 96-well plates and incubated with
0.00002 pM–25 nM of IFNa2, anti-CD20-IFNa2, or antiCD138-IFNa2 at 37C for 3 or 7 days. Relative metabolic
activity was determined using MTS solution (Promega,
Madison, WI) by measuring absorbance at 490 nm using a
Synergy HT Multi-Detection Microplate Reader (BioTek Instruments, Inc., Winooski, VT) with untreated cells being
100%. The GraphPad Prism (GraphPad Software, Inc., La
Jolla, CA) was used to analyze data by nonlinear regression
with the log (inhibitor) versus the response with a variable
slope. Data are expressed as a percentage of maximum metabolic activity. The experiments were performed in triplicate.
Apoptosis assay
Cells were treated with varying concentrations of IFNa2,
anti-CD20-IFNa2, anti-CD138 IgG, anti-CD138-IFNa2 of
ANTI-CD138-IFNa2 THERAPY AGAINST MULTIPLE MYELOMA
anti-CD138-IFNa2YNS for 2 or 3 days at 37C. Cells were
stained with Alexa Fluor 488-labeled Annexin V and propidium iodide (PI) using the Vybrant Apoptosis Kit #2
(Molecular Probes, Carlsbad, CA) as per the manufacturer’s
instructions and analyzed by flow cytometry.
Cells were treated for 2 days at 37C with 500 pM antiCD138-IFNa2 in the presence or absence of 1 nM rapamycin or 20 mM LY294002. Apoptosis was monitored by
staining with Alexa Fluor 488-labeled Annexin V and PI
and analyzed by flow cytometry as described above.
Caspase inhibition
U266 cells were treated for 2 days with 0.032 pM–
2.5 nM of anti-CD138-IFNa2 in the presence or absence of
50 mM zVAD-fmk (Calbiochem, San Diego, CA), a pancaspase inhibitor. Cell viability was measured using the
CellTiter Glo assay (Promega), as per the manufacturer’s
instructions.
Detection of IRF4
U266 cells were treated for 48 h with 1 nM of IFNa2,
anti-CD138-IFNa2, or anti-CD138-IFNa2YNS. Cells were
lysed using the RIPA buffer (50 mM Tris-HCl pH 8,
150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1%
SDS) containing a protease inhibitor cocktail (Roche Applied Science, Indianapolis, IN). The cytosolic fractions
were reduced with b-mercaptoethanol and separated by
SDS-PAGE. Following transfer to nitrocellulose membrane (Whatman, Piscataway, NJ) and blocking with 3%
bovine serum albumin (BSA) in phosphate buffered saline
(PBS) + 0.1% Tween 20, samples were incubated with
rabbit anti-IRF4 (Epitomics, Berlingham, CA) and rabbit
anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH;
Sigma-Aldrich). Secondary anti-rabbit IgG-HRP (GE Healthcare, Billerica, MA) was used and the blots were developed
using enhanced chemiluminescence (ECL; Thermo Fisher
Scientific). Films were imaged using a MultiImage Light
Cabinet (Alpha Innotech Corp., San Leandro, CA) and analyzed using the NIH ImageJ. Bands were normalized to a
GAPDH loading control and expressed as percent band intensity of untreated cells. The average from 3 different experiments is shown.
Detection of STAT1 and STAT3
U266 cells were treated for 0.5, 24 and 48 h with 100 pM
of IFNa2, anti-CD138-IFNa2, or anti-CD138-IFNa2YNS.
Cells were lysed using 50 mM Tris–HCl, pH 7.4, 1% NP-40,
150 mM NaCl, 1 mM EDTA, 1% glycerol, protease inhibitor
cocktail, and PhosSTOP (Roche Applied Science). The cytosolic fractions were reduced with b-mercaptoethanol and
separated by SDS-PAGE. Following transfer to PVDF
membrane (Boehringer-Mannheim, Germany) and blocking
in 3% BSA in PBS + 0.1% Tween 20, the blot was incubated
with primary antibody overnight at 4C. Secondary antirabbit IgG-HRP (GE Healthcare) was used and the blots
were developed using Western Lightning-ECL (PerkinElmer, Waltham, MA). Images were captured using a LAS1000 from Fujifilm. The following primary antibodies (Cell
Signaling Technology, Danvers, MA) were used: rabbit
polyclonal serum against phosphorylated STAT1-Y701,
283
STAT1, phosphorylated STAT3-Y705, and STAT3. Rabbit
anti-GAPDH was from Sigma-Aldrich. Quantitations of
bands are shown in Supplementary Table S1 (Supplementary Data are available online at www.liebertpub.com/jir).
Treatment of primary myeloma cells from patients
Patients with active myeloma were biopsied while off
therapy and myeloma cells isolated by negative antibody
selection to > 95% purity. Cells were incubated with 25 or
100 nM of anti-CD138 IgG, anti-CD138-IFNa2, or antiCD138-IFNa2YNS for 72 h. Percent viable cell recovery was
determined by Trypan Blue staining, with untreated control
cells designated as 100%.
In vivo anti-tumor activity against U266 cells
Six to 8 week old female NOD-scid IL2rcnull (NSG) mice
were used to establish U266 tumors. Mice were inoculated
subcutaneously with 1 · 107 cells at the base of the tail.
Mice were treated intravenously with PBS or 100 mg of antiCD138, anti-DNS-IFNa2, anti-CD138-IFNa2, anti-CD20IFNa2YNS, or anti-CD138-IFNa2YNS on days 14, 16, and 18
post tumor challenge. One group of mice received additional
treatments with anti-CD138-IFNa2YNS on days 24, 31, and
62 for a total of 6 treatments. Each group consisted of 8
mice. Bidirectional tumor growth was measured throughout
the experiment and mice were sacrificed when tumors
reached 1.5 cm as per the institutional guidelines. All animal
studies were performed in compliance with the US Department of Health and Human Services Guide for the Care
and Use of Laboratory Animals and were approved by the
UCLA Animal Research Committee.
Results
Production and characterization of anti-CD138IFNa2 and anti-CD138-IFNa2YNS fusion proteins
The aim of this study was to test the effectiveness of
using antibodies specific for CD138 to target IFN to MM
cells. The approach was to genetically fuse IFN to the end of
the CH3 domain of human IgG1 (Fig. 1A) containing the V
regions from the anti-CD138 antibody B-B4 (Wijdenes and
others 1996). For the initial experiments we elected to target
IFNa2, which has been used successfully in the treatment of
MM in the clinic, or a mutant higher affinity IFNa2YNS (see
‘‘Increasing the affinity of IFNa2 for its receptor increases
its potency in vitro’’ section).
Anti-CD138 IgG1 either unfused or fused to human IFNa2
was expressed in stable CHO transfectants. Purified proteins
were characterized with respect to their size and assembly
status. The purified fusion protein possess heavy (H) and light
(L) chains of the appropriate molecular weight and assemble
into complete H2L2 molecules (Fig. 1B) that bound CD138
expressed on the surface of HMCLs (data not shown).
IFNa2 fusion proteins inhibit the growth
of HMCLs with targeting by anti-CD138
further improving efficacy
Several different assays were used to determine whether
targeted fusion protein had cytoreductive activity against
HMCLs. To determine if fusion of IFNa2 to IgG had any
284
YOO ET AL.
with varying concentrations of anti-CD138, IFNa2, antiCD20-IFNa2, and anti-CD138-IFNa2 and then stained with
Alexa Fluor 488-labeled Annexin V and PI and examined by
flow cytometry (Fig. 2C). Unfused anti-CD138, which did not
inhibit cell viability (data not shown), also did not induce
apoptosis (Fig. 2C). Anti-CD138-IFNa2, anti-CD20-IFNa2,
and IFNa2 all showed a concentration-dependent increase
in the percentage of Annexin V + /PI - and Annexin V + /PI +
cells. However, cells treated with the targeted anti-CD138IFNa2 fusion protein showed the greatest percentage of Annexin V + cells, consistent with targeted anti-CD138-IFNa2
being more effective than untargeted anti-CD20-IFNa2 in
inhibiting cell proliferation (Fig. 2A). Following the shorter
2 day treatment, we observed both Annexin V + /PI - and
Annexin V + /PI + cells whereas by day 3, almost all Annexin
V + cells were also PI + (Fig. 2B).
Signaling pathways involved
in anti-CD138-IFNa2 activity
FIG. 1. Production of IFNa2 fusion proteins. (A) Schematic
diagram of anti-CD138 fusion proteins containing IFNa2 or
IFNa2YNS. The fusion proteins contain V regions of murine
antibody B-B4, which is specific for CD138, and human
IgG1,k constant regions. Human IFNa2 or IFNa2YNS is fused
to the C-terminus of the antibody through a SerGly4Ser peptide
linker. (B) SDS-PAGE analysis of reduced and unreduced
purified fusion proteins and control IgG. H, heavy; IFN, interferon; L, light.
effect on its activity, we compared the activity of IFNa2
alone to untargeted anti-CD20-IFNa2 fusion protein since
MM cells do not express CD20. HMCLs were treated with
varying concentrations for 3 days (U266, NCI-H929, MM1144) or 7 days (ANBL-6). Measurement of cellular metabolic
activity using the MTS assay showed that the anti-CD20IFNa2 fusion protein had similar activity as IFNa2 (Fig. 2A).
To ascertain the effect of targeting, we compared the activity
of the untargeted anti-CD20-IFNa2 fusion protein to that of
targeted anti-CD138-IFNa2 in a separate experiment. U266,
ANBL-6, NCI-H929, and MM1-144 cells were treated with
varying concentrations of untargeted and targeted fusion
protein for 3 days. Targeted anti-CD138-IFNa2 was significantly more potent than untargeted IFNa2 (Fig. 2A) with
*100-fold lower IC50 than untargeted IFNa2 in several
different assays.
Since we observed a loss in cell viability in response to
fusion protein treatment, we next wanted to determine if
apoptosis was being induced. U266, ANBL-6, NCI-H929,
and MM1-144 cells were treated for 3 days and stained
using Alexa Fluor 488-labeled Annexin V and PI and examined by flow cytometry (Fig. 2B). Anti-CD138-IFNa2
induced apoptosis in all 4 HMCLs. To further examine the
induction of apoptosis, U266 cells were incubated for 2 days
Many different signaling pathways have been shown to be
involved in IFNa-mediated toxicity. One important pathway
is the activation of caspases. Previous studies have shown
that induction of apoptosis following treatment with IFNa
was associated with activation of caspases-1, -2, -3, -8, -9 in
U266 cells (Thyrell and others 2002). To determine if caspases play a role in anti-CD138-IFNa2 toxicity against
U266, we treated the cells in the presence or absence of the
pan-caspase inhibitor, zVAD-fmk. Cells were incubated
with increasing concentrations of anti-CD138-IFNa2 with
or without 50 mM of zVAD-fmk and cell viability was
measured using the CellTiter Glo assay. In the presence of
zVAD-fmk, some of the cytotoxic effects of anti-CD138IFNa2 were decreased, but not eliminated (Fig. 3A). These
data confirm previous findings that activation of caspases
plays a role in IFNa-mediated cytotoxicity.
Thyrell and others (2004) reported that induction of apoptosis by IFNa in U266 cells involves the phosphoinositide
3 kinase (PI3K)/mammalian target of rapamycin (mTOR)
pathway. To determine if anti-CD138-IFNa2 acts similarly
through this pathway, we analyzed the effects of inhibitors
of PI3K/mTOR on the induction of apoptosis. Rapamycin
blocks mTOR whereas LY294002 blocks PI3K and to a
lesser extent mTOR (Brunn and others 1996). Cells were
treated with anti-CD138-IFNa2 for 2 days in the presence or
absence of the inhibitors. Apoptosis was monitored by
staining with Alexa Fluor 488-labeled Annexin V and PI and
analyzed by flow cytometry. Neither LY294002 nor rapamycin had any effects by itself (Fig. 3B, C, respectively).
When the cells were treated with anti-CD138-IFNa2, there
was an increase in Annexin V + cells. However, this induction
of apoptosis by the fusion protein was decreased in the
presence of LY294002 (Fig. 3B) and rapamycin (Fig. 3C).
Therefore, it appears that our fusion protein, like IFNa, utilizes the PI3K/mTOR pathway to induce apoptosis in the
U266 cell line.
Increasing the affinity of IFNa2 for its receptor
increases its potency in vitro
IFNa and IFNb are type I IFNs that bind to the same
receptor, which is composed of 2 transmembrane proteins,
IFNAR1 and IFNAR2. The IFNs differ in that IFNb has a
ANTI-CD138-IFNa2 THERAPY AGAINST MULTIPLE MYELOMA
285
FIG. 2. Growth inhibition
and induction of apoptosis by
untargeted and targeted IFNa2
fusion protein. (A) HMCLs
were incubated with varying
concentrations (0.00002 pM–
1 nM) of IFNa2 or anti-CD20IFNa2 for 3 days, except
ANBL-6 which was treated for
7 days (top panel). In a separate experiment, HMCLs were
incubated with (0.03 pM–
25 nM) of anti-CD20-IFNa2
or anti-CD138-IFNa2 for 3
days (bottom panel). Cellular
metabolic activity was measured using the MTS assay.
The experiment was performed in triplicate for each
concentration. (B) HMCLs
were untreated or treated with
500 pM of anti-CD138-IFNa2
for 3 days. Cells were stained
with Alexa Fluor 488-labeled
Annexin V and PI and analyzed by flow cytometry to
assess induction of apoptosis.
The number in the upper right
quadrants indicates the percentage of Annexin V + /PI +
cells. (C) U266 cells were
incubated with 0.5, 5, 50, or
500 pM of IFNa2, anti-CD20IFNa2, anti-CD138-IFNa2, or
anti-CD138 for 2 days and
analyzed as described above.
HMCLs, human myeloma cell
lines; PI, propidium iodide.
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FIG. 3. The antiproliferative effects of IFNa2 fusion proteins include the activation of caspases and the PI3K/mTOR
pathways. (A) U266 cells were treated for 2 days with
0.032 pM–2.5 nM of anti-CD138-IFNa2 in the presence or
absence of 50 mM of the pan-caspase inhibitor zVAD-fmk.
Cell viability was measured using the CellTiter Glo assay.
U266 cells were treated for 2 days with 500 pM of antiCD138-IFNa2 in the presence or absence of 20 mM LY294002
(B) or 1 nM rapamycin (C). Apoptosis was monitored by
staining with Alexa Fluor 488-labeled Annexin V and PI and
analyzed by flow cytometry. The number in the upper right
quadrants indicates the percentage of Annexin V + /PI + cells.
The number in the lower right quadrants indicates the percentage of Annexin V + /PI - cells. PI3K/mTOR, phosphoinositide 3 kinase/mammalian target of rapamycin.
YOO ET AL.
20- to 50-fold greater affinity for IFNAR1 than IFNa2
(Lamken and others 2004; Jaitin and others 2006; Jaks and
others 2007). This greater affinity has been shown to correlate with a significantly higher antiproliferative activity
against some malignancies ( Jaitin and others 2006; Kalie
and others 2007), making IFNb an attractive candidate for
cancer therapy. However, when human IFNb was genetically fused to IgG, we found that IFNb activity was reduced
by > 100-fold (data not shown). Therefore, we chose an
alternative approach to produce a high affinity mutant
IFNa2 fusion protein. This mutant contains mutations at 3
positions, H57Y/E58N/Q61S (IFNa2YNS), which confer a
60-fold increased affinity for IFNAR1 and a large increase
in antiproliferative activity compared to wild-type IFNa2
(Kalie and others 2007). Using the same approach as described above, we produced anti-CD138-IFNa2YNS in CHO
cells (Fig. 1) to compare the activity of the higher affinity
fusion protein to that of wild-type fusion protein. The ability
of the 2 fusion proteins to induce apoptosis was compared
by treating cells with anti-CD138-IFNa2 or anti-CD138IFNa2YNS for 2 or 3 days. Cells were then stained with
Alexa Fluor 488-labeled Annexin V and PI and analyzed by
flow cytometry. For the U266 and NCI-H929 cell lines,
treatment with anti-CD138-IFNa2YNS resulted in increased
levels of apoptosis compared to cells treated with anti-CD138IFNa2 (Fig. 4A). In addition, these cell lines showed greater
growth inhibition when they were treated with anti-CD138IFNa2YNS than when they were treated with anti-CD138IFNa2 and analyzed by the MTS assay (data not shown).
Similar results were seen in several other HMCLs (data not
shown). Disruption of the PI3K pathway using inhibitors resulted in decreased apoptotic activity against U266 following
treatment with anti-CD138-IFNa2YNS (data not shown) similar to what was seen following treatment with anti-CD138IFNa2 (Fig. 3).
To further explore the increased potency of anti-CD138IFNa2YNS, we tested for the expression and activation of
STAT proteins, which are known to be activated by IFNa.
U266 cells were treated with 100 pM of IFNa2, anti-CD138,
anti-CD138-IFNa2, or anti-CD138-IFNa2YNS for 0.5, 24,
and 48 h, and cell lysates were examined for levels of
STAT1, pSTAT1, STAT3, and pSTAT3 by western blotting.
The housekeeping gene GAPDH was used to control for gel
loading. Treatment with anti-CD138 did not activate STAT1
or STAT3. In contrast, pSTAT1 was seen 0.5 h following
treatment with IFNa2, anti-CD138-IFNa2, and anti-CD138IFNa2YNS. However, following treatment with anti-CD138IFNa2YNS, but not IFNa2 or anti-CD138-IFNa2, STAT1
phosphorylation persisted and strong pSTAT1 signal was
observed at 48 h. Similar results were seen for pSTAT3. All
treatments except anti-CD138 resulted in increased accumulation of STAT1 over time; increased accumulation of
STAT3 was not observed (Fig. 4B and Supplementary Table
S1). These data suggest that the increased potency of the
higher affinity anti-CD138-IFNa2YNS may be due at least, in
part, to the increased strength of the signal and/or the ability
to prolong signaling.
IFNa2 induces changes in IRF4 expression
in U266 cells
IRF4 is a transcription factor that is important in the
development of myeloid, lymphoid, and dendritic cells.
ANTI-CD138-IFNa2 THERAPY AGAINST MULTIPLE MYELOMA
287
FIG. 4. Anti-CD138-IFNa2YNS has greater activity
than anti-CD138-IFNa2. (A)
Cells were treated with 500 pM
of anti-CD138-IFNa2 or antiCD138-IFNa2YNS for 2 (U266)
or 3 (NCI-H929) days. Cells
were stained with Alexa Fluor
488-labeled Annexin V and PI
and analyzed by flow cytometry to assess induction of apoptosis. The number in the upper
right quadrants indicates the
percentage of Annexin V + /PI +
cells. The number in the lower
right quadrants indicates the
percentage of Annexin V + /PI cells. (B) U266 cells were
treated with 100 pM of the indicated proteins for 0.5, 24, or
48 h. Cell lysates were used
to determine the levels of
pSTAT1, total STAT1 protein,
pSTAT3, and total STAT3
protein by western blotting.
Protein loading was monitored
by probing the same membranes with anti-GAPDH.
IRF4 has also been shown to play a critical role in MM.
Some MMs have been shown to contain a chromosomal
translocation that juxtaposes the Ig H chain to the IRF4
locus, resulting in the overexpression of IRF4 (Iida and
others 1997; Yoshida and others 1999). However, the expression of IRF4 has been shown to be required for survival
of MM cells regardless of their genetic etiology even in
patient cells and MM cell lines that do not have genetic
alterations to the IRF4 locus (Shaffer and others 2008). IRF4
appears to be a master regulator in MM, controlling an
aberrant gene-expression program in this disease. Knockdown of IRF4 results in cell death because IRF4 targets
numerous genes that have a critical role in proliferation and
survival of MM cells (Shaffer and others 2009). To determine if IFNa and fusion protein treatment can cause changes to IRF4 expression, U266 cells were treated for 48 h
with IFNa2, anti-CD138-IFNa2, or anti-CD138-IFNa2YNS,
cell lysates prepared, and IRF4 levels determined by western blotting. A representative blot is shown in Fig. 5. Protein
levels were standardized against GAPDH. The average
quantitation from 3 independent experiments is shown. Interestingly, the level of IRF4 protein decreased when U266
cells were treated with IFNa2 or with fusion proteins, suggesting that this decrease may also play a role in the cytotoxic effects of IFNa2 against U266 cells.
Fusion proteins are effective against primary
MM cells from patients
To determine if the fusion proteins are effective against
primary tumors, patient cells were treated with anti-CD138,
anti-CD138-IFNa2, or anti-CD138-IFNa2YNS for 72 h and
the percentage of viable recovered cells compared to untreated cells was determined by Trypan Blue exclusion. As
expected, untreated primary cells demonstrated no increase
in cell number during this time as freshly obtained primary
MM cells do not proliferate ex vivo. Not surprisingly, there
was some variability in response among the 7 patients’ cells.
When the proteins were tested at 25 nM (Fig. 6A), antiCD138, anti-CD138-IFNa2, and IFNa2 (data not shown) had
little effect. In contrast, anti-CD138-IFNa2YNS treatment
resulted in significant decreases to cell viability when compared to anti-CD138 (P < 0.0001) or to anti-CD138-IFNa2
(P = 0.0009). Although anti-CD138-IFNa2 had little effect at
25 nM, when the protein was tested at 100 nM, anti-CD138IFNa2 was able to significantly reduce cell viability when
compared to anti-CD138 (P = 0.0026; Fig. 6B). These data
show that although both fusion proteins can affect cell viability, fusion with the higher affinity IFNa2YNS is more effective at lower concentrations against primary patient cells.
In a few primary samples where sufficient cell numbers were
288
YOO ET AL.
A
FIG. 5. IFNa2 and fusion proteins downregulate IRF4 in
U266 cells. Cells were treated for 48 h with 1 nM of IFNa2,
anti-CD138-IFNa2, or anti-CD138-IFNa2YNS. Cell lysates
were used to determine the levels of IRF4 protein by western
blotting. A representative blot is shown. The bands from the
blots were quantified using the NIH ImageJ software with
levels of GAPDH as a standard. The average of the quantitations from 3 different experiments is shown in the bar
graph. IRF4, IFN regulatory factor 4.
available to perform apoptosis assays, fusion proteins appeared to induce apoptotic death (data not shown).
Fusion proteins are effective in a murine
xenograft model of MM
To determine if the fusion proteins are protective against
MM in vivo, we used the xenograft model of U266 tumors in
NSG mice. The effect of the targeted fusion proteins (antiCD138-IFNa2 and anti-CD138-IFNa2YNS) was compared
to that of several controls. One group was not treated (PBS
only), whereas another group was treated with unfused antiCD138 IgG, which lacks IFNa2 activity. To determine the
effects of an untargeted IFNa2 fusion protein, mice were
treated with a fusion protein with an irrelevant specificity for
the hapten dansyl (anti-DNS-IFNa2). To determine the effects of an untargeted IFNa2YNS fusion protein, mice were
treated with anti-CD20-IFNa2YNS. Anti-CD20-IFNa2YNS
does not target myeloma cells since it is specific for human
CD20 and HMCLs do not express CD20.
U266 tumors were established in NSG mice, which are
severely immunocompromised, lacking mature T and B cells,
functional NK cells, and are deficient in cytokine signaling.
Mice were treated on days 14, 16, and 18 for all treatments.
For one of the treatment groups, anti-CD138-IFNa2YNS was
administered 3 additional times on days 24, 31, and 62 for a
total of 6 treatments. When survival and tumor size were
monitored, all treatment groups showed significant improve-
B
FIG. 6. Fusion proteins are effective against primary
myeloma cells. (A) Purified MM cells from 7 patients were
incubated with 25 nM of the indicated proteins for 72 h. Cell
viability was determined by Trypan Blue staining by comparing the number of recovered viable cells to that of untreated control cells. (B) Purified MM cells from 7 patients
were incubated with 100 nM of the indicated proteins for
72 h. Cell viability was assessed as described above. MM,
multiple myeloma.
ments in survival when compared with the PBS control group
(P £ 0.0003; Fig. 7A). Untargeted IFNa2 fusion proteins
conferred some level of protection as did anti-CD138 IgG
even though it showed no growth inhibitory activity in vitro
(Fig. 2C). However, treatment with targeted fusion protein
showed the best protection; anti-CD138-IFNa2 was more
effective than anti-DNS-IFNa2 (P < 0.0001) and anti-CD138IFNa2YNS was more effective than anti-CD20-IFNa2YNS
(P < 0.0001). Although anti-CD138-IFNa2YNS was more effective than anti-CD138-IFNa2 in in vitro assays using U266,
the 2 proteins had similar protective effects in vivo (P = 0.05).
The group receiving 6 doses of anti-CD138-IFNa2YNS
showed the greatest protection, suggesting that prolonged
treatment can enhance survival.
Discussion
IFNa therapy has been used for the treatment of MM,
but disagreement exists as to its efficacy. However, metaanalysis of 17 trials, including 2,333 patients who received
combination IFNa-chemotherapy or chemotherapy alone
showed significantly superior outcomes in IFNa-treated patients for relapse-free and overall survival; similarly, metaanalyses of maintenance treatments also showed significantly
better outcomes in the IFNa treatment arms than in untreated
controls (Fritz and Ludwig 2000), underscoring the fact that
IFNa can be an effective therapeutic against MM. Some of
the major problems for IFNa therapy are systemic toxicity
and short in vivo half-life. Our approach to circumventing
these problems was to fuse IFNa2 to anti-CD138 IgG1 to
increase its half-life and, by targeting, deliver an effective
ANTI-CD138-IFNa2 THERAPY AGAINST MULTIPLE MYELOMA
289
FIG. 7. Fusion proteins confer protection to mice in a xenograft model of MM. NSG mice were injected subcutaneously
with 1 · 107 U226 cells and treated on days 14, 16, and 18 as indicated by the black arrows with 100 mg of the indicated
proteins. One group received additional treatments on days 24, 31, and 62 as indicated by the red arrows. Survival and
tumor growth were monitored. Eight mice were treated for each group. P values were calculated between groups. - P ‡ 0.05,
*P £ 0.0003.
dose of IFNa2 to the tumor site without systemic side effects. We have previously successfully used this approach in
the treatment of human and murine B cell lymphomas using
human IFNa2- and murine IFNb-antibody fusion proteins,
respectively (Xuan and others 2010; Trinh and others 2013).
In this current study, we used the same approach to target
CD138 on MM cells. Anti-CD138-IFNa2 was more potent
than untargeted anti-CD20-IFNa2 at inhibiting the growth
of HMCLs, indicating that targeting enhances efficacy. The
activity of the fusion protein was partially dependent on
caspase activation as treatment with zVAD-fmk reduced
cytotoxicity. Anti-CD138-IFNa2 induced apoptosis of U266
cells in a concentration-dependent manner. The proapoptotic activity of the fusion protein dependent on activation of
the PI3K/mTOR pathway as treatment with rapamycin or
LY294002 led to decreases in the percentage of apoptotic
cells. Previous studies had demonstrated that IFNa-induced
apoptosis of U266 was dependent on activation of the PI3K/
mTOR pathway (Thyrell and others 2004) and a subset of
the genes upregulated by IFNa depends on an active PI3K
signaling pathway (Hjortsberg and others 2007). Interestingly, this IFNa-induced apoptosis requiring an active PI3K/
mTOR pathway was found to occur in the absence of
de novo transcription (Panaretakis and others 2008).
MM is characterized by significant heterogeneity. Studies
have shown that not all MM cells are responsive to treatment
with IFNa (Crowder and others 2005; Gomez-Benito and
others 2005) and responsiveness of HMCLs does not always
correlate with the level of IFNAR expression (Gomez-Benito
and others 2005). Recently, we showed that anti-CD20murine IFNb (mIFNb) is more effective than untargeted
mIFNb fusion protein and anti-CD20-murine IFNa (mIFNa)
against B cell lymphoma in a murine model (Trinh and
others 2013). Interestingly, while anti-CD20-mIFNa showed
only modest protection against tumors expressing low levels
of IFNAR, anti-CD20-mIFNb was able to significantly
prolong survival with some animals remaining tumor-free.
These data suggest that IFNs with higher affinity for IFNAR
may be better anticancer therapeutics. Therefore, we produced anti-CD138-IFNa2YNS, a mutant designed to mimic
IFNb, which has a higher affinity for IFNAR1 and greater
activity than IFNa2. When the efficacy of anti-CD138IFNa2YNS was compared to that of anti-CD138-IFNa2, we
found that anti-CD138-IFNa2YNS was more potent than the
wild-type fusion protein in HMCLs and in MM patient cells.
Treatment of U266 cells with anti-CD138-IFNa2YNS resulted in increased and prolonged activation of STAT1 as
compared with IFNa2 and anti-CD138-IFNa2 treatment,
290
suggesting that this may in part explain the increased potency of anti-CD138-IFNa2YNS.
Expression of IRF4 is associated with many lymphoid
malignancies. IRF4 acts as a master regulator of an aberrant,
malignancy-specific regulatory network, which influences
metabolism, membrane biogenesis, cell cycle progression,
cell death, and transcriptional regulation in myeloma cells
(Verdelli and others 2009). IRF4 inhibition has been found
to be toxic to myeloma cell lines regardless of the transforming oncogenic mechanism and downregulation by as
little as 50% can result in MM cell death (Shaffer and others
2008). IRF4 differs from other IRF family members in that
it is not induced by IFNs. Therefore, it is of great interest
that treatment with IFNa2, anti-CD138-IFNa2, and antiCD138-IFNa2YNS resulted in decreased expression of IRF4
in U266 cells. However, downregulation of IRF4 may not be
a general mechanism for fusion protein efficacy against
MM as we did not observe decreased IRF4 levels in 3 other
HMCLs treated with anti-CD138-IFNa2 or anti-CD138IFNa2YNS (unpublished observations). Nevertheless, it would
be interesting to expand upon these studies to elucidate the
mechanism by which IFNa2 downregulates IRF4 expression
in U266. Decreases in IRF4 does not affect cell cycle progression in SKMM1 cells (Shaffer and others 2008), and we
found this to be the case for U266 (data not shown).
Knockdown of IRF4 expression by RNA interference in
MM has been reported to result in nonapoptotic cell death
(Shaffer and others 2009). So in addition to proapoptotic
activity, IFNa fusion proteins may have other antiproliferative
effects.
One of the major challenges in translational research is to
determine if in vitro assays are predictive of in vivo outcome. In the case of IFNa2 fusion proteins, targeting improved the efficacy of IFNa treatment both in vitro and
in vivo. Anti-CD138-IFNa2 was more effective than untargeted anti-DNS-IFNa2 and anti-CD138-IFNa2YNS was
more effective than untargeted anti-CD20-IFNa2YNS against
U266 tumors in NSG mice. In addition, increasing the
treatment regimen from 3 to 6 doses also improved survival.
Some interesting observations emerged from the in vivo
studies. One was that treatment with anti-CD138-IFNa2 and
anti-CD138-IFNa2YNS showed similar levels of protection
in mice. This is in contrast to what was observed in our
in vitro studies in which the higher affinity anti-CD138IFNa2YNS was more effective than anti-CD138-IFNa2
against U266 cells in the MTS and the apoptosis assays.
However, when we tested for the induction of apoptosis at
low concentration (1 pM) of fusion proteins, we found that
anti-CD138-IFNa2 and anti-CD138-IFNa2YNS had comparable ability to induce apoptosis (data not shown). One explanation for the discrepancy between the in vivo and
in vitro data may be that the lower concentration more accurately reflects the in vivo situation. In addition, we found
that although anti-CD138 did not display antitumor activity
in vitro (Figs. 2 and 6 and data not shown), anti-CD138
provided a significant level of protection in mice and was
more protective than untargeted IFNa2 fusion proteins.
These data suggest that the antitumor activity may be
achieved partly through the effector functions of IgG, which
would be observable in vivo, but not in vitro. Although NSG
mice are severely immunocompromised, they do contain
functional monocytes and neutrophils (Racki and others
2010), which may be involved in tumor killing through
YOO ET AL.
antibody-dependent cell-mediated cytotoxicity (ADCC)
(Ravetch and Kinet 1991). Therefore, the fusion proteins
may prove to be even more effective in the treatment of
human patients since the immunomodulatory activities of
human IFNa2 and effector functions such as complementdependent cytotoxcity and ADCC associated with the human
IgG Fc region are not fully functioning in mice.
Our studies have shown that targeting of IFNa2 and
higher affinity IFNa2YNS through the anti-CD138 moiety
can be an effective strategy in the treatment of MM. Fusion
of IFNa2 and IFNa2YNS to anti-CD138 should increase their
half-life and decrease the systemic side effects of IFNa2,
making for an effective therapeutic against MM.
Acknowledgments
This work was supported by the Senior Research Award
(S.L.M.), the Dean Assink/Multiple Myeloma Research
Foundation Senior Research Award (S.L.M.), the National
Institutes of Health (grants 2RO1CA111448, 1RO1CA132778
and 1R21CA168491 to A.L.), and grants from the Multiple
Myeloma Research Foundation (A.L.), and the Veteran’s
Administration (A.L.).
Author Disclosure Statement
No competing financial interests exist.
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Address correspondence to:
Dr. Sherie L. Morrison
Department of Microbiology, Immunology
and Molecular Genetics
University of California Los Angeles
247 BSRB
615 Charles E. Young Drive East
Los Angeles, CA 90095
E-mail: sheriem@microbio.ucla.edu
Received 24 July 2014/Accepted 18 September 2014