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LYMPHOID NEOPLASIA
Targeted delivery of interferon-alpha via fusion to anti-CD20 results in potent
antitumor activity against B-cell lymphoma
Caiyun Xuan,1 Kristopher K. Steward,2,3 John M. Timmerman,2,3 and Sherie L. Morrison1,2
1Department
of Microbiology, Immunology and Molecular Genetics, 2Molecular Biology Institute, and 3Division of Hematology and Oncology, Department of
Medicine, University of California Los Angeles
The anti-CD20 antibody rituximab has
substantially improved outcomes in patients with B-cell non-Hodgkin lymphomas. However, many patients are not
cured by rituximab-based therapies, and
overcoming de novo or acquired rituximab resistance remains an important
challenge to successful treatment of Bcell malignancies. Interferon-alpha (IFN␣)
has potent immunostimulatory properties and antiproliferative effects against
some B-cell cancers, but its clinical utility
is limited by systemic toxicity. To improve
the efficacy of CD20-targeted therapy, we
constructed fusion proteins consisting of
anti-CD20 and murine or human IFN␣.
Fusion proteins had reduced IFN␣ activity in vitro compared with native IFN␣, but
CD20 targeting permitted efficient antiproliferative and proapoptotic effects against
an aggressive rituximab-insensitive human CD20ⴙ murine lymphoma (38C13huCD20) and a human B-cell lymphoma
(Daudi). In vivo efficacy was demonstrated against established 38C13huCD20 grown in syngeneic immunocom-
petent mice and large, established Daudi
xenografts grown in nude mice. Optimal
tumor eradication required CD20 targeting, with 87% of mice cured of rituximabinsensitive tumors. Gene knockdown
studies revealed that tumor eradication
required expression of type I IFN receptors on the tumor cell surface. Targeting
type I IFNs to sites of B-cell lymphoma by
fusion to anti-CD20 antibodies represents a potentially useful strategy for
treatment of B-cell malignancies. (Blood.
2010;115(14):2864-2871)
Introduction
The anti-CD20 antibody rituximab (C2B8/Rituxan; Genentech/
Biogen-IDEC) has substantially improved treatment outcomes in
B-cell non-Hodgkin lymphomas (NHLs), achieving high response
rates in low-grade B-cell lymphomas,1 and improving survival in
both indolent and aggressive lymphomas in combination with
chemotherapy.2,3 However, many tumors do not respond to or
relapse after rituximab-based therapies.4 Thus, new approaches are
needed to improve anti-CD20 efficacy and overcome rituximab
resistance.
The in vivo antilymphoma effects of rituximab are believed to
be mediated by antibody dependent cell-mediated cytotoxicity
(ADCC), complement-dependent cytotoxicity (CDC), induction of
apoptosis in tumor cells, and recruitment of T cells responding to
tumor antigens released upon antibody-mediated tumor lysis.5-7
Clinical studies have suggested that ADCC plays a dominant role in
rituximab action in humans.8,9 Thus, attempts have been made to
boost rituximab-mediated ADCC by activation of Fc receptor–
bearing natural killer (NK) cells, monocytes/macrophages, or
granulocytes via systemic administration of cytokines such as
interleukin-2, interleukin-12, or granulocyte-macrophage colonystimulating factor,10-12 with limited efficacy. None of these trials
involving systemic administration of cytokines offered a clear
advantage over the expected efficacy of rituximab alone, likely due
to the inability of systemically administered agents to achieve high
concentrations within the tumor bed.
Interferon-alpha (IFN␣), a member of the type I interferon
family (␣, ␤, ␻), is a pleiotropic cytokine with attractive features
for combination with rituximab in treating NHL.13,14 Beneficial
properties of IFN␣ against NHL and other cancers include direct
antiproliferative and proapoptotic effects,15-17 blockade of autocrine growth factor loops,18 repression of c-myc oncogene expression,19 down-regulation of telomerase activity,20 and inhibition of
angiogenesis.21 Favorable immunologic effects of IFN␣ for lymphoma treatment include activation of T cell, NK cell, and dendritic
cell functions, as well as up-regulation of class I major histocompatibility complex and CD20 molecules on the tumor cell surface.22,23
The single-agent clinical activity of IFN␣ against NHL has been
demonstrated in numerous early clinical trials,24-26 and in more
recent series by Armitage and Coiffier27 and Armitage et al.28
Clinical studies combining systemic IFN␣ therapy with rituximab
suggest benefits of addition of the cytokine.29,30 However, despite
its potent antitumor properties, the clinical utility of IFN␣ in cancer
therapy has been severely limited by the substantial toxicities
associated with systemic administration.31 Contributing to this
failure is the short serum half-life of IFN␣ (5 hours), and the lack of
effective levels of the cytokine within tumor sites. Pharmacokinetic
studies have indicated that only 0.01% of subcutaneously injected
IFN␣ reaches the target tumor site.32 Given these limitations, it is
difficult to achieve effective IFN␣ concentrations at sites of
malignant disease without causing systemic toxicity.
The limitations of systemic IFN␣ therapy have led to the
exploration of alternative strategies to deliver IFN␣ safely and
effectively into the tumor vicinity. Reports have shown that
intratumoral delivery of IFN␣ by direct injection can lead to
durable complete tumor regressions of NHL.33,34 Tumor-specific
IFN␣ delivery via transduced monocytes or adenoviral vectors is
Submitted October 23, 2009; accepted January 15, 2010. Prepublished online
as Blood First Edition paper, February 4, 2010; DOI 10.1182/blood-2009-10250555.
payment. Therefore, and solely to indicate this fact, this article is hereby
marked ‘‘advertisement’’ in accordance with 18 USC section 1734.
The publication costs of this article were defrayed in part by page charge
© 2010 by The American Society of Hematology
2864
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BLOOD, 8 APRIL 2010 䡠 VOLUME 115, NUMBER 14
also effective in inhibition of tumor growth and angiogenesis in
glioma and metastatic carcinoma tumor models.35,36 Despite promising efficacy in mouse models, clinical use of viral vectors in
humans is problematic. An attractive alternative approach to
improving efficacy while reducing toxicity is to deliver IFN␣ via
an antibody fusion protein. Fusion proteins have been used
extensively to deliver cytokines, radioisotopes, and toxins for
cancer therapy.37 Antibody-cytokine fusion proteins use the unique
targeting ability of antibodies to guide antitumor agents specifically
into the tumor vicinity where these drugs can work most effectively
to eradicate tumor cells. In the case of B-cell lymphoma, tumorspecific targeting of IFN␣ via anti-CD20 may represent a viable
2-pronged therapy.
We previously reported that an anti-HER2/neu antibody–IFN␣
fusion protein had in vitro and in vivo efficacy against an
experimental murine tumor overexpressing HER2/neu.38 We now
report the production and characterization of anti–CD20-IFN␣
fusion proteins carrying either murine or human IFN␣.
Methods
Cell lines
HEK293T cells were purchased from ATCC and grown in Iscove modified
Dulbecco medium (Invitrogen) supplemented with 2mM L-glutamine,
50␮M ␤-mercaptoethanol, and 5% calf serum. The 38C13-huCD20 cells,
which express human CD20, were previously described.39 Both 38C13 and
38C13-huCD20 were cultured in RPMI 1640 (Invitrogen) supplemented
with 2mM L-glutamine, 50␮M ␤-mercaptoethanol, and 10% fetal bovine
serum. The cell line 38C13-huCD20 IFN␣ receptor knockdown (IFNAR
KD) was produced by transducing 38C13-huCD20 cells with a lentiviral
vector encoding an shRNA targeting the IFNAR1 subunit of IFNAR with
the sense sequence 5⬘-GCGTCTACATTATAGATGACAA-3⬘ as previously
described.40 Briefly, 38C13-huCD20 cells were infected at 0.3 multiplicity
of infection and sorted for high green fluorescent protein fluorescence using
fluorescence activated cell sorting (FACS). Single-cell clones were isolated
by limiting dilution. Daudi cells were purchased from ATCC and grown in
the same RPMI 1640 supplemented as described.
Vectors
Heavy chain and light chain variable regions of the anti–human CD20
monoclonal antibody 2B8, the murine parent of rituximab, were provided
by Dr Anna Wu (University of California Los Angeles)41 and cloned into
human ␥3 heavy chain (pAH6180) and ␬ light chain (pAG3551) expression
vectors, respectively, to produce anti–CD20-immunoglobulin G3 (IgG3).
To construct anti–CD20-IgG3-mIFN␣, polymerase chain reaction (PCR)
was used to introduce a BamHI restriction enzyme site upstream and an
XbaI restriction enzyme site downstream of the mature murine IFN␣1 gene
amplified by PCR from genomic DNA of BALB/c mice with the forward
primer 5⬘-CGCGGATCCTGTGACCTGCCTCAGACTC-3⬘ and the reverse primer 5⬘-GCTCTAGATCATTTCTCTTCTCTCAGTCTTC-3⬘. The
PCR product was ligated into the vector pAH9612 containing an IgG3
constant region with an anti-CD20 heavy chain variable region and a
GGGGS peptide linker at the end of CH3. To construct anti–CD20-IgG1hIFN␣, PCR was used to introduce a BamHI restriction enzyme site
upstream and an XbaI restriction enzyme site downstream of the mature
human IFN␣2 gene amplified by PCR from human genomic DNA with the
forward primer 5⬘-CGCGGATCCTGTGATCTGCCTCAAACCCA-3⬘ and
the reverse primer 5⬘-GCTCTAGATCATTCCTTACTTCTTAAACTTTC3⬘. The PCR product was ligated into the vector pAH10812 containing an
IgG1 constant region with an anti-CD20 heavy chain variable region and a
GGGGS peptide linker at the end of CH3.
ANTI–CD20-IFN␣ FOR B-CELL LYMPHOMA THERAPY
2865
Protein production and purification
Heavy and light chain vectors were transiently transfected into HEK293T
cells via polyethylenimine transfection42 and supernatants collected every
other day for 2 weeks. Cell-free culture supernatants were passed through a
protein A Sepharose 4B fast flow (Sigma-Aldrich) column and bound
protein was eluted with 0.1M glycine, pH 2.5. Eluted fractions were
neutralized immediately with 2M Tris-HCl, pH 8.0. Fractions were run on
sodium dodecyl sulfate–polyacrylamide gel electrophoresis gels and stained
with Coomassie blue to verify protein purity and integrity. Concentrations
of proteins were determined using a standard bicinchoninic acid (BCA)
assay (Pierce).
Flow cytometry
The 38C13-huCD20 cells (106) were incubated with 1 ␮g of anti–CD20mIFN␣, anti–DNS-IFN␣, rituximab (Genentech Inc), or isotype control
antibody (Sigma-Aldrich) for 1 hour at 4°C in FACS buffer (phosphatebuffered saline ⫹ 1% bovine serum albumin ⫹ 0.1% NaN3). Binding of
fusion protein was detected using biotinylated rat anti–human ␬, followed
by streptavidin–phycoerythrin (PE; BD Biosciences). IFNAR expression
was detected using a biotinylated anti–mouse IFNAR antibody (clone
MAR1-5A3; Leinco Technologies) followed by streptavidin-PE. Data were
acquired using a FACScan flow cytometer (Becton Dickinson) and
analyzed using FlowJo software (TreeStar Inc).
Inhibition of proliferation assay
The 38C13-huCD20 cells were incubated with various treatments at 37°C
for 48 hours. Cell viability was quantified using MTS (3-(4,5 dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl-2-(4-sulfophenyl)-2H-tetrazolium)
solution (Promega) by measuring absorbance at 490 nm using a Synergy
HT Microplate Reader. Data were analyzed using KC4 software (Bio-Tek).
Percentage of inhibition of proliferation was calculated as [1 ⫺ (ODexp/
ODuntreated)] ⫻ 100. Activity of IFN␣ was compared at the dosage required
for 50% growth inhibition based on the best fit curve. Recombinant murine
IFN␣ reference standard was obtained from the National Institutes of
Health.
Apoptosis assay
The 38C13-huCD20 or Daudi cells were incubated with various treatments
at 37°C for 48 or 72 hours, respectively. Cells were stained with
annexinV–fluorescein isothiocyanate (FITC) and propidium iodide (PI) to
distinguish populations of early apoptotic (annexinV⫹/PI⫺), late apoptotic
(annexinV⫹/PI⫹), and necrotic (annexinV⫺/PI⫹) cells using Vybrant Apoptosis Kit #2 (Molecular Probes). The percentage of apoptotic cells was
calculated as the sum of the percentages of early apoptotic cells and late
apoptotic cells.
In vivo antitumor activity against murine B-cell lymphoma
Female (6-8 weeks old) C3Hf/Sed/Kam mice were bred and housed at the
University of California Los Angeles (UCLA) Defined Pathogen Colony
according to institutional guidelines. All animal procedures were approved
by the UCLA Animal Research Committee. 38C13-huCD20 was thawed
3 days before tumor challenge and split the day before use. For tumor
challenge, cells were washed twice in Hanks balanced salt solution (HBSS)
and diluted to the appropriate concentration in HBSS. Challenge inocula
consisted of 5 ⫻ 103 38C13-huCD20 cells injected subcutaneously above
the base of the tail. Experimental groups were treated with 10 ␮g of
anti–CD20-mIFN␣ fusion protein intravenously 1, 2, and 3 or 5, 6, and
7 days after tumor challenge. In some experiments, fusion protein–treated
mice received additional 30-␮g doses 12 and 19 days after tumor challenge.
In some cases, mice surviving initial tumor challenge were rechallenged
subcutaneously at day 60 with 5 ⫻ 103 parental 38C13 cells. Mice were
followed for survival and killed when tumors reached 1.4 cm in diameter.
Bidirectional tumor growth measurements were taken throughout the
experiments.
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2866
XUAN et al
In vivo antitumor activity against human B-cell lymphoma
Female nude mice (6-8 weeks old) were bred and housed as described for
C3Hf/Sed/Kam mice. Challenge inocula consisted of 1.2 ⫻ 107 Daudi cells
resuspended in a total of 200 ␮L of HBSS, injected subcutaneously above
the base of the tail. Mice (32% of total number inoculated) with established
tumors greater than 0.5 cm in diameter after 30 days were randomly
assigned to treatment groups. Groups were treated intravenously with either
30 ␮g of anti–CD20-hIFN␣ fusion protein, equimolar amounts of rituximab (20 ␮g), or HBSS 3 times at weekly intervals beginning 30 days after
tumor challenge. Tumor volume was calculated using a modified ellipsoid
formula43 where tumor volume ⫽ 0.52 ⫻ (length ⫻ width2).
Statistical analysis
Apoptosis data were compared using the unpaired, 2-tailed Student t test.
Survival differences among groups of mice were assessed using the
Kaplan-Meier method with the log-rank test using Prism software (GraphPad Software). P values were considered statistically significant at P less
than .05.
BLOOD, 8 APRIL 2010 䡠 VOLUME 115, NUMBER 14
2B8,44 so that our recombinant fusion proteins could be directly
compared with this prototypical anti-CD20 therapeutic. To generate the fusion proteins, the N-terminus of either mature murine
IFN␣1 (mIFN␣) or mature human IFN␣2a (hIFN␣) was fused via a
Gly4Ser linker to the C-terminus of the heavy chain of anti-CD20
(Figure 1A). Nontargeted anti–DNS-mIFN␣ (antidansyl) and anti–
DNS-hIFN␣ were also produced, along with unfused anti-CD20
IgG3. Proteins were purified using protein A Sepharose and eluted
fractions analyzed by sodium dodecyl sulfate–polyacrylamide gel
electrophoresis. Under nonreducing conditions, the molecular
weight of anti–CD20-mIFN␣ was approximately 210 kDa (data
not shown), indicating proper assembly of an H2L2 molecule. After
treatment with ␤-mercaptoethanol, the fusion protein migrated as
heavy chains attached to murine IFN␣ (⬃ 80 kDa) and light chains
(⬃ 25 kDa; data not shown). Similar results were seen with
anti–CD20-hIFN␣ (data not shown).
Anti–CD20-mIFN␣ and anti–CD20-hIFN␣ retain their
antigen-binding abilities
Results
Production and characterization of anti–CD20-mIFN␣ and
anti–CD20-hIFN␣
We chose to construct anti–CD20-IFN␣ fusion proteins using the
variable regions from the rituximab parent monoclonal antibody
The binding of the fusion proteins to human CD20 was assessed by
flow cytometry against the human CD20–expressing tumor lines
38C13-huCD20 and Daudi. Anti–CD20-mIFN␣ bound 38C13huCD20 (Figure 1B) in a manner similar to that of native
rituximab. Anti–CD20-hIFN␣ bound Daudi, a human B-cell lymphoma cell line (data not shown). These data show that C-terminal
Figure 1. Production and characterization of anti–CD20-mIFN␣. (A) The heavy chain and light chain variable regions of anti-CD20 2B8 were cloned into human ␥3 heavy
chain and human ␬ light chain expression vectors. Mature murine IFN␣ was inserted downstream of a Gly4Ser (GlySer) linker following the CH3 domain of the constant region
gene. (B) Flow cytometry using 38C13-huCD20 cells demonstrates that the fusion protein retains the ability to bind human CD20. Cells were incubated with anti–CD20-mIFN␣
(black peak), rituximab (solid line), isotype control for IgG1 (dashed line), or isotype control for IgG3 (shaded peak). Anti–human kappa-PE was used to detect cell-bound
antibodies. (C) Comparative biologic activity of IFN␣ fusion protein and recombinant mIFN␣ as measured by inhibition of growth of wild-type 38C13 (hu-CD20 negative) cells.
(D) Antiproliferative activity of fusion protein against 38C13-huCD20 cells. Cells were incubated with the indicated proteins for 48 hours. Cell proliferation was measured using
the MTS assay and the data are represented as mean ⫾ SD of triplicate values.
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BLOOD, 8 APRIL 2010 䡠 VOLUME 115, NUMBER 14
Figure 2. Anti–CD20-mIFN␣ potently induces apoptosis of 38C13-huCD20 cells
in a dose-dependent manner. 38C13-huCD20 cells were incubated with various
concentrations of the indicated proteins for 48 hours. Staining with annexinV-FITC
and PI was performed to distinguish necrotic (annexin⫺PI⫹), early apoptotic
(annexin⫹PI⫺), and late apoptotic (annexin⫹PI⫹) cell populations. The percentage of
total apoptotic cells was quantified for each sample as the sum of early apoptotic and
late apoptotic cells (bottom panel). Experiments were performed in triplicate, and
error bars indicate mean ⫾ SD. *P ⫽ .001. **P ⬍ .001.
fusion of IFN␣ to anti-CD20 did not affect the ability of the
antibody to bind its antigen.
In vitro targeting of IFN␣ via anti-CD20 significantly enhances
its efficacy against human CD20ⴙ murine B-cell lymphoma
38C13-huCD20
To quantify the IFN␣ bioactivity of the anti–CD20-mIFN␣ fusion
protein, MTS assays measuring cell viability were performed on
non–CD20-expressing parental 38C13 cells, which are sensitive to
growth inhibition by IFN␣.45 Anti–CD20-mIFN␣ and anti–DNSmIFN␣ had equivalent ability to inhibit the proliferation of 38C13;
however, their activities were 300-fold reduced compared with
recombinant mIFN␣ (Figure 1C). In contrast, anti–CD20-mIFN␣
had 105-fold higher antiproliferative activity than nontargeted
anti–DNS-mIFN␣ against 38C13 cells expressing human CD20,39
indicating that targeting of IFN␣ markedly enhances its efficacy
(Figure 1D). Compared with recombinant murine IFN␣ (rmIFN␣),
anti–CD20-mIFN␣ had 103-fold higher antiproliferative activity
against 38C13-huCD20 despite its reduced IFN␣ activity (Figure
1D). Over an equivalent range of concentrations (from 1 to
100pM), native rituximab did not exhibit any antiproliferative
effects against this tumor (data not shown). When proapoptotic
activity was assayed in 38C13-huCD20 after 48 hours of treatment
with 100pM concentrations of fusion proteins, anti–CD20-mIFN␣
induced significantly higher levels of apoptosis (50.3% annexinVpositive cells) than equivalent amounts of the nontargeted anti–DNSmIFN␣ (9.7%), and the combination of anti–DNS-mIFN␣ ⫹
rituximab (14.3%; P ⫽ .001; Figure 2). A similar trend was
observed at a lower concentration of anti–CD20-mIFN␣ (10pM),
with apoptosis induced in 30.7% of the treated cells, whereas the
other treatments caused significantly less apoptosis. Taken together, these data show that targeting of IFN␣ was essential for the
efficacy of this fusion protein because the combination of rituximab and a nontargeted IFN␣ fusion protein was significantly less
effective than anti–CD20-mIFN␣.
Anti–CD20-mIFN␣ prevents growth of 38C13-huCD20 tumors in
vivo without apparent systemic toxicity
We next chose to evaluate our anti–CD20-mIFN␣ in vivo using an
immunocompetent, syngeneic mouse model, in which the species
of the fused mIFN␣ moiety matched that of the tumor host,
permitting evaluation of potential IFN toxicity. The 38C13huCD20 tumor line, which expresses physiologic levels of human
CD20, is a model of rituximab-insensitive lymphoma when
ANTI–CD20-IFN␣ FOR B-CELL LYMPHOMA THERAPY
2867
injected subcutaneously into C3H mice. After 3 or more days of
establishment, 38C13-huCD20 tumors cannot be eradicated, even
by a large dose (500 ␮g) of single-agent rituximab.39 Initial
experiments in this model demonstrated that an early treatment
regimen consisting of 0.4-␮g, 2-␮g, or 10-␮g doses of anti–CD20mIFN␣ given 1, 2, and 3 days after tumor inoculation could
eradicate tumors from 50%, 75%, and 100% of the animals,
respectively (P ⱕ .01 for all groups vs HBSS treated), illustrating
potent, dose-dependent activity of the fusion protein in vivo
(Figure 3A). The anti–CD20-mIFN␣ fusion protein was also
effective at treating more established tumors (Figure 3B). When
5-day established tumors were treated with three 10-␮g doses of
anti–CD20-mIFN␣ given 5, 6, and 7 days after tumor inoculation,
the overall survival of the fusion protein–treated mice was higher
compared with HBSS-treated mice (P ⬍ .001) and mice treated
with a combination of anti-CD20 and rmIFN␣ given at equivalent
molar doses (P ⫽ .007; Figure 3B). Consistent with our in vitro
results, targeting of the fusion protein played a significant role in its
efficacy given the improved overall survival of the anti–CD20mIFN␣ group versus the nontargeted anti–DNS-mIFN␣ group
(P ⬍ .001). In this treatment model, rituximab failed to prolong
survival of any animals. Indications of systemic toxicity such as
lethargy, ruffled fur, or weight loss were not observed in any of the
animals.
Repeat dosing with anti–CD20-mIFN␣ efficiently eradicates
established tumors
Although a single round of CD20-targeted IFN␣ significantly
prolonged the survival of mice, some animals eventually developed
tumors. To test whether efficacy could be improved by repeat
dosing, 5-day established 38C13-huCD20 tumors were treated with
either a single round of anti–CD20-mIFN␣ or with a repeat dosing
schedule that included 2 additional doses of 30 ␮g of fusion protein
Figure 3. Anti–CD20-mIFN␣ treatment can eradicate established, rituximabinsensitive human CD20ⴙ B-cell lymphoma. (A) Early treatment model. Mice
(n ⫽ 4 per group) were treated with the indicated doses of fusion protein or saline
(HBSS) 1, 2, and 3 days after tumor inoculation (arrows). *P ⫽ .01. (B) Late treatment
model. Mice (n ⫽ 8 per group) were treated with 10 ␮g of fusion protein or equivalent
molar concentration of the indicated proteins 5, 6, and 7 days after tumor inoculation
(arrows). Mice in the repeat dosing group were given 2 additional 30-␮g doses of
fusion protein 12 and 19 days after tumor inoculation (thick arrows). *P ⫽ .001.
**P ⫽ .007. The data from 3 independent experiments, each of which included HBSS
and fusion protein groups, were combined.
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XUAN et al
BLOOD, 8 APRIL 2010 䡠 VOLUME 115, NUMBER 14
with 38C13-huCD20 (mean fluorescence intensity ⫽ 20). Knockdown of IFNAR did not affect CD20 expression as determined by
flow cytometry (data not shown), and the in vivo growth kinetics of
38C13-huCD20 IFNAR KD were similar to those of 38C13huCD20 (data not shown). In vitro apoptosis studies showed that
38C13-huCD20 IFNAR KD had decreased sensitivity to fusion
protein treatment. At 48 hours after treatment with 1000pM
anti–CD20-mIFN␣, 58.2% of parental 38C13-huCD20 cells were
apoptotic compared with only 13.7% of the 38C13-huCD20
IFNAR KD cells (Figure 4B). In animal studies, the treatment
regimen that had previously been effective against 5-day established 38C13-huCD20 tumors strikingly failed to delay or prevent
tumor onset in mice inoculated with 38C13-huCD20 IFNAR
(Figure 4C). Thus, IFNAR expression at the tumor cell surface is
required for anti–CD20-mIFN␣–mediated activity in vivo, suggesting that direct tumor cytotoxicity is the primary mechanism of
action of the fusion protein in this model.
Anti–CD20-hIFN␣ is effective against the human B-cell
lymphoma Daudi
Figure 4. Antitumor efficacy of anti–CD20-mIFN␣ requires IFNAR expression
on tumor cells. (A) Flow cytometric analysis of IFNAR expression on indicated cell
lines. 38C13-huCD20 transduced with IFNAR-specific shRNA (38C13-huCD20
IFNAR KD), 38C13-huCD20 transduced with nonspecific shRNA (38C13-huCD20
control), and 38C13-CD20 parental cells were stained with anti–IFNAR-biotin primary
antibody (clone MAR1-5A3) and detected with streptavidin-PE. Biotinylated IgG1
isotype–stained control is also shown. (B) Apoptosis assay using parental 38C13huCD20 and 38C13-huCD20 IFNAR KD. Cells were treated with 1000pM anti–CD20mIFN␣ and stained with annexinV-FITC and PI 48 hours later. Flow cytometry
patterns and gate frequencies in percentages are shown in the upper right hand
corner of the annexin⫹ gates. (C) Effective in vivo treatment of 38C13-huCD20 with
anti–CD20-mIFN␣ requires IFNAR expression on tumor cells. Mice (n ⫽ 8 per group)
were treated 5, 6, and 7 days after tumor inoculation with 10 ␮g of anti–CD20-mIFN␣
or the molar equivalent of the indicated proteins. Mice were followed for survival and
killed when tumors reached 1.4 cm in diameter as per institutional guidelines. Mice
treated with HBSS were used as control.
12 and 19 days after tumor inoculation (Figure 3B). At the time of
administration of the first repeat dose on day 12, tumors were
palpable in the HBSS-treated mice, but not in the fusion protein–
treated animals. Whereas the single round of therapy with fusion
protein eradicated tumors from 29% of mice, the 3 weekly rounds
of therapy cured 87% of animals (P ⬍ .001 vs HBSS, P ⫽ .001 vs
single round of therapy). No animals showed any signs of toxicity
during therapy, highlighting the safety of this approach in immunocompetent hosts.
We next sought to confirm the efficacy of our anti–CD20-IFN␣
fusion protein strategy against human B-cell lymphoma. We
generated a human IFN␣ fusion protein, anti–CD20-hIFN␣, and
compared its antitumor activity against the human B-cell lymphoma line Daudi with that of rituximab, nontargeted anti–DNShIFN␣, and a combination of anti–DNS-hIFN␣ and rituximab.
Addition of 100pM rituximab to Daudi cells did not induce
significant apoptosis (5.0%) compared with untreated cells (5.0%;
data not shown; Figure 5). In contrast, cells treated with 100pM
anti–CD20-hIFN␣ were significantly more apoptotic (31.7%) than
rituximab-treated cells (5.0%, P ⬍ .001). Targeting was important
for fusion protein activity as equimolar amounts of anti–DNShIFN␣ induced significantly lower levels of apoptosis (15.7%)
compared with anti–CD20-hIFN␣ (P ⫽ .001). Anti–CD20-hIFN␣
was also more effective at inducing apoptosis than the combination
of rituximab and anti–DNS-hIFN␣ (P ⫽ .001). When the treatment
dose was decreased to 10pM, similar results were obtained, with
anti–CD20-hIFN␣ inducing significantly more apoptosis than the
combination of anti–DNS-hIFN␣ and rituximab (P ⫽ .003). Consistent with the results obtained in our studies with the murine
IFN␣ fusion protein, anti–CD20-hIFN␣ has potent activity against
human lymphoma cells that is dependent on targeting of IFN␣ to
the tumor cell surface.
Fusion protein efficacy requires IFNAR expression on tumor
cells
IFN␣ can act on tumor cells directly by inducing apoptosis upon
binding to its receptor IFNAR on the cell surface, or indirectly by
recruiting host immune cells such as NK cells into the tumor
microenvironment to promote tumor killing.13 To distinguish
between these possibilities, we used shRNA to generate 38C13huCD20 IFNAR KD, a cell line with decreased expression of
IFNAR (Figure 4A; mean fluorescence intensity ⫽ 11) compared
Figure 5. Anti–CD20-hIFN␣ fusion protein has proapoptotic activity against the
human B-cell lymphoma Daudi. (A) Daudi lymphoma cells were incubated with
various concentrations of the indicated proteins for 72 hours. Staining with annexinVFITC and PI was performed to distinguish necrotic (annexin⫺PI⫹), early apoptotic
(annexin⫹PI⫺), and late apoptotic (annexin⫹PI⫹) cell populations. The percentage of
total apoptotic cells was quantified for each sample as the sum of early apoptotic and
late apoptotic cells. Experiments were performed in triplicate, and error bars indicate
mean ⫾ SD. *P ⫽ .001. **P ⫽ .003.
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BLOOD, 8 APRIL 2010 䡠 VOLUME 115, NUMBER 14
Anti–CD20-hIFN␣ can cure well-established human lymphoma
xenograft tumors
To extend our in vitro results against human lymphoma cells to an
in vivo setting, we used a xenograft model of established Daudi
tumors in nude mice. Treatment with 3 weekly 30-␮g doses of
anti–CD20-hIFN␣, equimolar doses of rituximab or HBSS were
initiated only after tumors reached at least 0.5 cm in diameter
(Figure 6A-C). In 7 of 7 mice treated with anti–CD20-hIFN␣, there
was rapid and uniform tumor regression. In comparison, only 2 of
5 mice treated with rituximab experienced tumor regression.
Spontaneous tumor regression was seen in 1 of 5 mice treated with
HBSS. The overall survival of the fusion protein–treated mice was
significantly better than that of mice treated with rituximab
(P ⫽ .02), and all fusion protein–treated mice remained tumor free
after 60 days (Figure 6D). In contrast, the rituximab-treated mice
did not exhibit enhanced survival compared with mice treated with
HBSS (P ⫽ .5).
Discussion
The use of systemically administered IFN␣ in treating B-cell
lymphoma has been problematic because of the toxicity of this
potent antitumor agent. In the present study, we demonstrated that
the targeting of IFN␣ to lymphomas by an antibody specific for the
B-cell differentiation antigen CD20 allows concentrations of
the cytokine to reach effective levels at the tumor site, leading to
the efficient cure of tumor-bearing mice in the absence of systemic
toxicity. The anti–CD20-IFN␣ fusion protein was significantly
more effective against CD20-expressing tumors than both singleagent rituximab and the combination of anti-CD20 and systemic
IFN␣. Thus, IFN␣, an agent limited in practice by its toxicity, has
here been exploited for its advantageous antitumor properties to
potent effect when targeted via anti-CD20.
ANTI–CD20-IFN␣ FOR B-CELL LYMPHOMA THERAPY
2869
In vitro treatment of lymphoma cells expressing human
CD20 with anti–CD20-IFN␣ induced higher levels of apoptosis
than did rituximab, a nonspecific antibody-IFN␣ fusion (anti–
DNS-IFN␣), or a combination of the two (Figures 2,5). This
finding predicted an advantage when treating tumors in vivo,
which was indeed the case. For our initial in vivo studies, we
chose the 38C13-huCD20 model, a syngeneic human CD20–
expressing murine B-cell lymphoma grown in immunocompetent C3H mice.39 The 38C13 lymphoma is a highly aggressive
tumor model in which mice develop micrometastases in the
spleen, lymph nodes, and bone marrow within 6 to 9 days of
subcutaneous tumor inoculation,46 thus representing a stringent
setting in which to evaluate novel therapeutics. This setting not
only allows recruitment of the full range of host innate and
adaptive immune effector mechanisms, but also assessment of
IFN␣ toxicity. In vivo treatment of mice bearing 38C13huCD20 tumors with anti–CD20-IFN␣ resulted in significant
delay in tumor growth and/or cure in small and established
tumors. The low dose of 10 ␮g of anti–CD20-mIFN␣ given on
3 consecutive days completely prevented growth of small
tumors when administered as an early treatment regimen
beginning 1 day after tumor inoculation. Treatment with equivalent doses of rituximab had no effect, consistent with the
antitumor activity being mediated through IFN␣ and not via
ligation of CD20. Indeed, earlier studies reported that 500 ␮g of
rituximab given as a single dose 1 day after tumor inoculation
cured only 38% of small tumors.39 In a treatment model of
established 38C13-huCD20 tumors, 3 daily 10-␮g doses of
anti–CD20-mIFN␣ administered in a late treatment regimen
beginning 5 days after tumor challenge resulted in significant
delay of tumor growth in all animals and 29% cure. Animals
treated with rituximab did not experience any delay in tumor
growth or enhancement in survival compared with HBSStreated mice. To improve upon the 29% cure rate of animals with
established tumors, mice were retreated at weekly intervals
Figure 6. Anti–CD20-hIFN␣ completely cures established human xenograft tumors. (A-C) Tumor growth in mice (n ⫽ 5-7 per group) inoculated subcutaneously with
Daudi cells and treated as indicated with 3 weekly doses of 30 ␮g of fusion protein, the equivalent molar concentration of rituximab, or HBSS. Treatment was administered 30,
37, and 44 days after tumor inoculation (arrows) to mice with tumors at least 0.5 cm in diameter. HBSS was injected as a control. Symbols represent individual mice.
(D) Survival curves for the mice whose tumor growth is shown in panels A-C. *P ⫽ .02.
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2870
BLOOD, 8 APRIL 2010 䡠 VOLUME 115, NUMBER 14
XUAN et al
when tumor cells surviving the initial treatment would have
proliferated significantly. This repeat treatment regimen resulted
in 87% cure with no observed toxicity. Thus, repeat administration of anti–CD20-mIFN␣ has the potential to completely cure
lymphoma with none of the associated systemic toxicity that has
been an obstacle in IFN␣-based cancer therapy. Tumor-specific
targeting of mIFN␣ was critically important in the action of
anti–CD20-mIFN␣ as the nontargeted anti–DNS-mIFN␣ fusion
protein did not prolong or promote survival. This demonstrates
that delivery of IFN␣ via anti-CD20 directly into the tumor
vicinity is superior to systemic IFN␣ administration and is a
viable approach to increasing the therapeutic index of this
potent antitumor agent. Because of the multifunctional nature of
the anti–CD20-IFN␣ fusion protein, the mechanism by which it
prevents tumor growth is of interest. In vivo experiments using
38C13-huCD20 IFNAR KD, a cell line with decreased IFNAR
expression, resulted in complete abrogation of fusion protein–
mediated tumor killing. These data suggest that the antitumor
effect of anti–CD20-mIFN␣ is mediated primarily and possibly
exclusively through the induction of tumor cell death via a direct
interaction between targeted IFN␣ and its receptor on the
surface of tumor cells.
ADCC,8,9 CDC,47 and direct induction of apoptosis48 have been
shown to contribute to the antitumor activities of rituximab.
However, in the present studies, ADCC and CDC do not appear to
play a significant role in fusion protein–mediated protection against
tumor growth, although their involvement cannot be excluded.
Given that in most cases only 10-␮g doses of fusion protein were
used in our in vivo studies, these results are not surprising. The low
doses of fusion protein used in treatments would not be expected to
saturate the CD20 molecules present on tumor cell surfaces. This
lack of saturation would limit activities such as ADCC and CDC,
which require binding of effector cells or complement, respectively, to closely spaced Fc regions of an antibody.
To extend our observations in 38C13-huCD20 to a human
tumor model, an anti-CD20 human IFN␣ fusion protein (anti–CD20hIFN␣) was generated. When its activity was evaluated using the
human lymphoma Daudi, consistent with what had been observed
in the murine tumor model, anti–CD20-hIFN␣ had far higher
proapoptotic activity than rituximab or the combination of rituximab and hIFN␣. Importantly, the fusion protein was effective at
very low doses where rituximab treatment did not induce significant levels of apoptosis. In a xenograft model of large, wellestablished Daudi tumors, low doses of fusion protein treatment
resulted in rapid regression of 100% of tumors. In marked contrast,
tumor regression was seen in only 40% of the mice treated with
equimolar concentrations of rituximab. Fusion protein–treated
mice also had enhanced survival compared with rituximab-treated
mice, and all were tumor-free 60 days after the initial tumor
challenge. These results indicate that the fusion protein can
eliminate bulky tumors and may be an effective therapeutic against
established human lymphoma.
Recently, Rossi et al reported a CD20-targeted tetrameric
human IFN␣ construct that showed efficacy against human
lymphoma xenografts in severe combined immunodeficient
mice in vivo.49 The molecule in this study was generated using a
“dock and lock” method whereby 4 molecules of IFN␣2b were
attached to an antibody targeting CD20. The resulting conjugates showed potent interferon activity comparable with that of
PEG-intron (Peginterferon alpha-2b). Although this construct
displayed promising activity against several human lymphoma
xenografts, its high-level interferon activity raises the concern
of systemic toxicity against nontumor cells that could not be
evaluated in xenograft models, as mouse cells are insensitive to
human IFN␣2b. In our studies, using a syngeneic immunocompetent murine model, no toxicity was observed with the
species-appropriate mouse IFN␣ fusion protein. Importantly,
the fusion protein demonstrated powerful efficacy against
lymphoma despite a reduction in its IFN␣ activity. Thus, a
targeted therapeutic in which the activity of the attached
interferon is reduced may have a higher therapeutic index.
Anti–CD20-hIFN␣ has the potential to be a versatile therapeutic. It could be administered in combination with rituximab and/or
chemotherapy to augment antitumor activity. As a monotherapy,
anti–CD20-hIFN␣ may provide a viable alternative to rituximab in
the clinic for the subpopulation of patients in whom rituximabrefractory tumors arise by unknown mechanisms. Various hypotheses such as loss or down-regulation of CD20 and the presence of
circulating CD20 have been proposed to explain how resistance to
rituximab arises. A study by Jazirehi et al50 reported that this
phenomenon was independent of diminished CD20 expression, but
in fact resulted from aberrant expression of signaling molecules or
dysregulation of signaling pathways. Because anti–CD20-IFN␣ is
essentially a 2-pronged attack whereby both CD20 and IFNAR
signaling pathways can be activated to induce tumor cell apoptosis,
it may remain efficacious in instances when CD20 signaling
pathways become dysregulated in patients with rituximab-resistant
tumors. It would be of interest to examine the effect of fusion
protein treatment on rituximab-refractory lymphomas in future
studies.
IFN␣ has potent biologic activities against B-cell malignancies,
but its clinical utility is currently limited by systemic toxicities. We
hypothesized that targeting IFN␣ directly to tumor cells via fusion
to an anti-CD20 antibody could be a valuable agent for treating
lymphomas. Our results indicate that anti–CD20-IFN␣ fusion
proteins possess a markedly improved therapeutic index, exhibiting
the ability to eradicate both murine and human lymphomas in vivo
using a small fraction of the IFN␣ that would be used in
conventional systemic dosing. Therefore, we propose that the
anti–CD20-hIFN␣ fusion protein warrants further evaluation as a
lymphoma therapeutic.
Acknowledgments
This work was supported by the National Institutes of Health (RO1
GM074051) and the Leukemia & Lymphoma Society Translational
Research Program (grant 6098). C.X. was supported by the UCLA
Dissertation Year Fellowship.
Authorship
Contribution: C.X. designed and performed experiments, analyzed
data, and wrote the paper; K.K.S. designed and performed experiments, reviewed data, and reviewed the paper; and J.M.T. and
S.L.M designed experiments, reviewed data, and wrote the paper.
Conflict-of-interest disclosure: The authors declare no competing financial interests.
Correspondence: Sherie L. Morrison, Professor of Microbiology, Immunology and Molecular Genetics, 247 BSRB, 615 Charles
E. Young Dr East, Los Angeles, CA 90095; e-mail: sheriem@
microbio.ucla.edu.
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BLOOD, 8 APRIL 2010 䡠 VOLUME 115, NUMBER 14
ANTI–CD20-IFN␣ FOR B-CELL LYMPHOMA THERAPY
2871
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From www.bloodjournal.org by guest on June 14, 2017. For personal use only.
2010 115: 2864-2871
doi:10.1182/blood-2009-10-250555 originally published
online February 4, 2010
Targeted delivery of interferon-alpha via fusion to anti-CD20 results in
potent antitumor activity against B-cell lymphoma
Caiyun Xuan, Kristopher K. Steward, John M. Timmerman and Sherie L. Morrison
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