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Chimeric CLL-1 Antibody Fusion Proteins Containing GranulocyteMacrophage Colony-Stimulating Factor or Interleukin-2 With Specificity for
B-Cell Malignancies Exhibit Enhanced Effector Functions While Retaining
Tumor Targeting Properties
By Jason L. Hornick, Leslie A. Khawli, Peisheng Hu, Maureen Lynch, Peter M. Anderson, and Alan L. Epstein
Although monoclonal antibody (MoAb) therapy of the human malignant lymphomas has shown success in clinical
trials, its full potential for the treatment of hematologic malignancies has yet to be realized. To expand the clinical potential of a promising human-mouse chimeric antihuman Bcell MoAb (chCLL-1) constructed using the variable domains
cloned from the murine Lym-2 (muLym-2) hybridoma, fusion
proteins containing granulocyte-macrophage colony-stimulating factor (GM-CSF) (chCLL-1/GM–CSF) or interleukin (IL)2 (chCLL-1/IL–2) were generated and evaluated for in vitro
cytotoxicity and in vivo tumor targeting. The glutamine synthetase gene amplification system was employed for high
level expression of the recombinant fusion proteins. Antigenic specificity was confirmed by a competition radioim-
munoassay against ARH-77 human myeloma cells. The activity of chCLL-1/GM–CSF was established by a colony
formation assay, and the bioactivity of chCLL-1/IL–2 was
confirmed by supporting the growth of an IL-2–dependent
T-cell line. Antibody-dependent cellular cytotoxicity against
ARH-77 target cells demonstrated that both fusion proteins
mediate enhanced tumor cell lysis by human mononuclear
cells. Finally, biodistribution and imaging studies in nude
mice bearing ARH-77 xenografts indicated that the fusion
proteins specifically target the tumors. These in vitro and in
vivo data suggest that chCLL-1/GM–CSF and chCLL-1/IL–2
have potential as immunotherapeutic reagents for the treatment of B-cell malignancies.
q 1997 by The American Society of Hematology.
W
cell lines in vitro and to improve the survival of human
lymphoma-bearing severe combined immunodeficiency
(SCID) mice by the induction of apoptosis (Funakoshi et al,
manuscript in preparation). In antibody-dependent cellular
cytotoxicity (ADCC) assays, however, murine Lym-2 mediates low tumor lysis with human mononuclear effector cells.
A human-mouse chimeric derivative designated chCLL-1
has therefore been constructed to increase its effector functions. To further enhance the immunotherapeutic potential
of this chimeric antibody for the treatment of B-cell malignancies, antibody fusion proteins containing human GMCSF and IL-2 have been generated. In this study, we describe
the effector functions mediated by these recombinant molecules and demonstrate their tumor targeting abilities in a
nude mouse xenograft model.
ITH THE EXCEPTION of a chimeric anti-CD20
monoclonal antibody (MoAb), which has produced
tumor regressions in patients with relapsed B-cell non-Hodgkin’s lymphoma (NHL),1 unconjugated MoAbs have demonstrated limited therapeutic responses.2 Radioimmunotherapy,
on the other hand, has shown considerable promise in clinical
studies, particularly in the treatment of B-cell NHL.3 The
efficacy of radioimmunotherapy is restricted, however, either
by dose-limiting thrombocytopenia or more severely by the
presence of bone marrow disease. In these settings, effective
therapy with unconjugated MoAbs would be desirable for
the induction of tumor remission. For this purpose, the combination of MoAbs and biologic response modifiers has been
investigated as a means of increasing tumor lysis. Cytokines
including interleukin-2 (IL-2) and granulocyte-macrophage
colony-stimulating factor (GM-CSF) have been shown to
enhance both in vitro cytotoxicity mediated by MoAbs
against tumor targets and in vivo killing of tumor xenografts
in animal model systems.4-9 Because of the toxicity of systemically administered cytokines, however, methods are
needed to target these biologically potent immunologic mediators to the tumor site. One approach, gene transfer, has
demonstrated that tumor cells engineered to secrete cytokines stimulate antitumor immunity and rejection in animal
models,10-17 illustrating the importance of localizing cytokines to tumors. However, at the present time, this approach
is impractical in the clinical setting. An alternative method
is the use of antibody-cytokine fusion proteins to direct such
immunologically active molecules to tumor sites.18-20 In this
way high local concentrations of cytokines within tumors can
be achieved and systemic toxicity is minimized or avoided.
In this report, we describe the development of such molecules for the treatment of hematologic malignancies. Lym2 is a murine IgG1 MoAb directed against a human major
histocompatability complex (MHC) class II variant that is
strongly reactive with a high percentage of human B-cell
NHL, chronic lymphocytic leukemia, and multiple myeloma
cell lines and biopsy specimens.21 Lym-2 has recently been
shown to have a direct inhibitory effect on human lymphoma
MATERIALS AND METHODS
Reagents
The plasmid pcD-hGM/Eo-CSF containing the human GM-CSF
cDNA22 was obtained from the American Type Culture Collection
From the Department of Pathology, University of Southern California School of Medicine, Los Angeles; the Division of HematologyOncology, the Department of Medicine, UCLA School of Medicine,
Los Angeles, CA; and the Section of Pediatric Hematology-Oncology, Mayo Clinic, Rochester, MN.
Submitted December 16, 1996; accepted January 31, 1997.
Supported in part by Cancer Therapeutics, Inc (Los Angeles, CA),
Techniclone Corp (Tustin, CA), Brilliance Pharmaceuticals (Shanghai, China), and a grant from Children’s Cancer Research Fund,
the Hedberg Foundation (Minneapolis, MN).
Address reprint requests to Alan L. Epstein, MD, PhD, Department of Pathology, University of Southern California School of Medicine, 2011 Zonal Ave, HMR 210, Los Angeles, CA 90033.
The publication costs of this article were defrayed in part by page
charge payment. This article must therefore be hereby marked
‘‘advertisement’’ in accordance with 18 U.S.C. section 1734 solely to
indicate this fact.
q 1997 by The American Society of Hematology.
0006-4971/97/8912-0001$3.00/0
Blood, Vol 89, No 12 (June 15), 1997: pp 4437-4447
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4438
HORNICK ET AL
(clone 57594; Rockville, MD). The plasmid pBC12/HIV/IL-2 containing the human IL-2 cDNA23 was obtained from the American
Type Culture Collection (clone 67618). The plasmids pEE6hCMVB and pEE12 were purchased with the Glutamine Synthetase Gene
Amplification System from Celltech Biologics (Slough, UK). Restriction endonucleases, T4 DNA ligase, and other molecular biology
reagents were purchased from New England Biolabs (Beverly, MA)
or Boehringer Mannheim (Indianapolis, IN). RPMI-1640 medium,
minimal essential medium (MEM) nonessential amino acids solution, penicillin-streptomycin solution, Dulbecco’s phosphate-buffered saline (PBS), dialyzed fetal bovine serum, Sephadex, buffer
salts, and other reagents such as chloramine T, sodium metabisulfite,
hydrogen peroxide, and ABTS (2,2*-azino–bis(3-ethylbenzthiazoline-6-sulfonic acid) diammonium salt) were purchased from Sigma
Chemical Co (St Louis, MO). Hybridoma-SFM medium with and
without glutamine was purchased from Life Technologies (Gaithersburg, MD). Fetal bovine serum was obtained from HyClone Laboratories, Inc (Logan, UT). Iodine-125 and iodine-131 were obtained
as sodium iodide in 0.1 N sodium hydroxide from DuPont/New
England Nuclear (North Billerica, MA). Balb/C and athymic nude
mice were purchased from Harlan Sprague Dawley (Indianapolis,
IN).
Antibodies and Cell Lines
The murine MoAb Lym-2 (muLym-2, IgG1), directed against a
B-cell surface antigen,21 was obtained from Techniclone International, Inc (Tustin, CA). The human-mouse chimeric MoAb Lym1 (chLym-1, IgG1k) was generated as previously described.24 The
chimeric MoAb CLL-1 (chCLL-1, IgG1k) was produced as previously described (Funakoshi et al, in preparation). The chimeric
MoAb TNT-1 (chTNT-1, IgG1k), the cDNAs for whose variable
regions were cloned from the murine TNT-1 hybridoma,25 was constructed and expressed in the same manner as chCLL-1. The murine
Lym-2 antiidiotype MoAb (7E2) was generated as previously described for the antiidiotype to Lym-1 (1A7).24 Iodine-125 and iodine131-labeled MoAbs were prepared using a modified chloramine T
method as previously described.24 The NS0 murine myeloma cell
line, which was obtained from Celltech Biologics, was grown in
nonselective medium consisting of Hybridoma-SFM supplemented
with 10% fetal bovine serum, L-glutamine, MEM nonessential amino
acids solution, penicillin G (100 U/mL), and streptomycin (100 mg/
mL). Selective medium consists of Hybridoma-SFM without glutamine supplemented with 10% dialyzed fetal bovine serum, glutamic
acid, asparagine, nucleosides, penicillin G, and streptomycin, according to the protocol provided with the Glutamine Synthetase
Gene Amplification System (Celltech Biologics). The ARH-77 human myeloma cell line,26 obtained from the American Type Culture
Collection, was grown in RPMI-1640 medium supplemented with
10% fetal bovine serum, L-glutamine, penicillin G, and streptomycin.
Construction of Expression Vectors
The expression vectors were constructed using standard techniques. The expression vector for chCLL-1, 12/chCLL-1/HL, was
used as the parent vector. This plasmid contains the cDNA sequences
for the human-mouse chimeric CLL-1 heavy and light chains, each
under the control of the cytomegalovirus (CMV) major immediate
early promoter, and the cDNA sequence for glutamine synthetase,
under the control of the SV40 early promoter. Two oligonucleotide
primers, 5*- GGTAAAGCGGCCGCAGGAGGTGGTAGCGCACCCGCCCGCTCGCCCAGC - 3* and 5* - TCAATGCGGCCGCTCACTCCTGGACTGGCTCCCAGCA - 3*, were used to amplify
by polymerase chain reaction (PCR) the human GM-CSF cDNA
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from the pcD-hGM/Eo–CSF plasmid template. To amplify the human IL-2 cDNA from the pBC12/HIV/IL-2 plasmid template, two
primers, 5* - GGTAAAGCGGCCGCAGGAGGTGGTAGCGCACCTACTTCAAGTTCTACA - 3* and 5* - TCATGCGGCCGCTCAAGTTAGTGTTGAGATGATGCT - 3*, were used. The PCR fragments were each inserted into the Not I site of 12/chCLL-1/HL,
resulting in the expression vectors 12/chCLL-1/HL/GM-CSF and
12/chCLL-1/HL/IL-2, encoding the chimeric light chain and a fusion
protein consisting of the chimeric CLL-1 heavy chain with human
GM-CSF or human IL-2 at its C-terminus.
Expression and Purification of Fusion Proteins
The fusion proteins were expressed from NS0 murine myeloma
cells according to the protocol of the manufacturer (Celltech Biologics). Briefly, linearized plasmids were electroporated into NS0 cells,
which were plated in nonselective Hybridoma-SFM medium. Selective glutamine-free medium was added 24 hours later. When
transfectants appeared approximately 3 weeks later, supernatants
were tested for the presence of chimeric fusion protein by indirect
enzyme-linked immunosorbent assay (ELISA). The highest-producing clones were identified by 24-hour rate of production assays. To
maximize the yield of chCLL-1/GM-CSF, amplification of vector
copy number was achieved by expanding the clone and incubating
the cells in increasing concentrations of methionine sulfoximine, a
specific inhibitor of glutamine synthetase. Three to 4 weeks later,
viable clones were again assayed for rate of chimeric fusion protein
production. After subcloning by limiting dilution, the highest-producing clones were expanded, incubated in 10 L bioreactors, and
chCLL-1/GM-CSF and chCLL-1/IL-2 were purified stepwise from
cell culture medium by protein A affinity chromatography and ionexchange chromatography, as described previously.24 The purity of
each fusion protein was examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) in a reducing gel according to the method of Laemmli27 and by high performance liquid
chromatography (HPLC). The samples were filtered through a 0.22
mm Nalgene disposable filter unit before injection. The fusion proteins were analyzed with a Beckman HPLC Gold System (Beckman
Instruments, Fullerton, CA) equipped with two 110B solvent pumps,
a 210A valve injector, a 166 programmable UV detector, and a
406 analog interface module. Size exclusion chromatography was
performed on a G4000SW column (TosoHaas; Montgomeryville,
PA) with 0.1 mol/L PBS, pH 7.2 as the solvent system, eluting at
a flow rate of 1 mL/min. The UV absorbance of the HPLC eluate
was detected at 280 nm.
Immunoassays
ELISA. Chimeric fusion protein-containing supernatants were
initially identified by indirect ELISA using murine Lym-2 antiidiotype 7E2 MoAb, as described previously.24 For production rate
assays, 106 cells were plated in 1 mL of selective medium and
allowed to incubate for 24 hours. ELISA was then performed as
before. Supernatants were serially diluted and applied to wells of
microtiter plates coated with goat antihuman IgG (H/L) (CalTag,
South San Francisco, CA). Dilutions of a control chimeric antibody
were used to generate a standard curve using 4-parameter fit by an
automated ELISA reader (Bio-Tek Instruments, Inc, Winooski, VT),
from which concentrations of unknowns were estimated. Rates of
production expressed as mg/mL/106 cells/24 hours were compared
to identify the highest producing clones.
ARH-77 cell competition radioimmunoassay. The antigen-binding activity of chCLL-1/GM-CSF and chCLL-1/IL-2 was determined
by a competition radioimmunoassay for binding to fixed ARH-77
myeloma cells. For these studies, 2 1 106 ARH-77 cells previously
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GM-CSF AND IL-2 ANTIBODY FUSION PROTEINS
4439
fixed in 2% paraformaldehyde28 were incubated with 20 ng of 125Ilabeled muLym-2 and serial dilutions of cold muLym-2, chCLL-1/
GM-CSF, chCLL-1/IL-2, or an irrelevant MoAb (chLym-1). The
cells and MoAbs or fusion proteins were incubated for 1 hour at
room temperature with constant mixing. The cells were then washed
twice, and the cell pellet-associated radioactivity was measured in
a gamma counter. Maximal binding was determined from tubes containing no cold antibodies.
Determination of Avidity
To determine the avidity constants of chCLL-1/GM-CSF and
chCLL-1/IL-2, a fixed cell radioimmunoassay was performed using
the method of Frankel and Gerhard.29 Each experimental variable
was run in duplicate. ARH-77 myeloma cell suspensions containing
106 cells/mL were incubated with 10 to 110 ng of 125I-labeled chCLL1/GM-CSF or chCLL-1/IL-2 in 200 mL PBS for 1 hour at room
temperature with constant mixing. The cells were then washed three
times with PBS containing 1% bovine serum albumin to remove
unbound antibody and counted in a gamma counter. The amount of
fusion protein bound was then determined by the remaining cellbound radioactivity (cpm) in each tube and the specific activity (cpm/
ng) of the radiolabeled fusion protein. Scatchard plot analysis was
used to obtain the slope. The equilibrium or avidity constant Ka was
calculated by the equation K Å 0(slope/n), where n is the valence
of the antibody (2 for IgG).
Isolation of Bone Marrow Cells
Bone marrow samples were obtained in preservative-free heparin
from normal donors after receiving their informed consent (with the
approval of UCLA Institutional Review Board). Cells were diluted
with an equal volume of PBS containing 0.6% ACD-A (anticoagulant citrate dextrose solution, Formula A; Baxter-Fenwal Corp, Deerfield, IL) and mononuclear cells (MNC) were isolated by gradient
centrifugation on Ficoll-Paque (Pharmacia LKB; Uppsala, Sweden)
followed by two washes with PBS/ACD-A. CD34/ cells were purified from MNC using a CD34/ Progenitor Cell Isolation Kit (Miltenyi Biotec; Auburn, CA) without modification of the manufacturer’s instructions.
Colony Assays
Bone marrow MNC (7.5 1 104 cells/well) or CD34/ cells (1 1
104 cells/well) were plated in triplicate in 24-well plates in semisolid
medium containing 0.3% bacto agar in Iscove’s Modified Dulbecco’s
Medium, 20% fetal bovine serum (Atlanta Biological, Norcross,
GA), 50 mg/mL gentamicin, 0.4 mmol/L L-glutamine. Colony assays
were supplemented with either hu-GM-CSF (generously provided
by Amgen; Thousand Oaks, CA), chCLL-1/GM-CSF, or chCLL-1.
Cultures were maintained humidified at 377C in 5% CO2. Colonies
containing more than 30 cells were enumerated after 14 to 16 days
in culture.
IL-2 Bioassay
Biologic activity of chCLL-1/IL-2 was determined by a standard
IL-2–dependent T-cell proliferation assay.30 Carrier-free recombinant IL-2 obtained from Hoffmann La Roche, Inc (Nutley, NJ) was
used as a standard. Roche IL-2 stock (7.8 mg/mL, specific activity
É12 1 106 IU/mg) was diluted to yield a stock solution containing
2 1 106 IU/mL. Growth of the IL-2–dependent murine T-cell line,
CTLL-2, was used to determine the amount of IL-2 bioactivity in a
sample. Briefly, serially diluted samples and standard were incubated
with 2 1 104 CTLL-2 cells in triplicate for 20 hours at 377C in 96well flat bottom microtiter plates. The cells were then pulsed with
0.5 mCi of 3H-thymidine for 6 hours, and the samples were harvested
and counted.
Cytotoxicity Assays
ADCC was performed using the CytoTox 96 Non-Radioactive
Cytotoxicity Assay (Promega, Madison, WI), which is a colorimetric
assay that quantitatively measures lactate dehydrogenase release.31
Effector cells were peripheral blood MNC or neutrophilic polymorphonuclear leukocytes (PMN). MNC were isolated from healthy
human donors by Ficoll-Paque gradient centrifugation, and PMN
were purified by centrifugation through a discontinuous percoll gradient (70% and 62%) followed by hypotonic lysis to remove residual
erythrocytes as described previously.32 ARH-77 myeloma cells were
used as target cells. ARH-77 cells were suspended in HybridomaSFM medium supplemented with 2% fetal bovine serum and plated
in 96-well V-bottom microtiter plates at 2 1 104 cells/well. Antibody
or fusion protein preparations (chCLL-1/GM-CSF, chCLL-1/IL-2,
chCLL-1, muLym-2, or chTNT-1 as an isotype-matched irrelevant
control) were added in triplicate to individual wells at 1 mg/mL, and
effector cells were added at various effector:target cell ratios (12.5:1
to 50:1). The plates were incubated for 4 hours at 377C, after which
the supernatants were harvested, lactate dehydrogenase release was
determined, and % specific lysis was calculated according to the
protocol of the manufacturer. Data are reported as mean { standard
deviation (SD). Differences between groups were analyzed by unpaired Student’s t-test.
Pharmacokinetic and Biodistribution Studies
Six-week-old Balb/C mice were used to determine the pharmacokinetic clearance of chCLL-1, chCLL-1/GM-CSF and chCLL-1/IL2. Groups of mice (n Å 5) were administered intraperitoneal (IP)
injections of 125I-labeled fusion proteins (30 to 40 mCi/mouse). The
whole body activity at injection and at selected times thereafter was
measured with a CRC-7 microdosimeter (Capintec, Inc, Pittsburgh,
Fig 1. Schematic diagram depicting the linker
containing the Not I cloning site between the human
g1 and human GM-CSF or human IL-2 cDNAs in the
chimeric CLL-1 heavy chain/cytokine fusion genes.
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HORNICK ET AL
Fig 2. Electrophoretic identification of chCLL-1/cytokine fusion
proteins. Coomassie Blue-stained 4% to 20% acrylamide gradient trisglycine reduced gel of purified chCLL-1 (lane 1), chCLL-1/GM-CSF
(lane 2), and chCLL-1/IL-2 (lane 3).
PA). The data were analyzed and half-lives were determined using
the RSTRIP pharmacokinetic program (MicroMath, Inc, Salt Lake
City, UT). To determine the tissue biodistribution of chCLL-1/GMCSF and chCLL-1/IL-2, 6-week-old female athymic nude mice were
irradiated with 400 rads from a cesium source, 3 days after which
they were injected with a 0.2 mL inoculum containing 4 1 107
ARH-77 cells and 4 1 106 human fetal lung fibroblast feeder cells
subcutaneously (SC) in the left thigh. The tumors were grown for
3 weeks until they reached approximately 1 cm in diameter. Within
each group (n Å 5), individual mice were injected intravenously
(IV) with a 0.1 mL inoculum containing 100 mCi/10 mg of 125Ilabeled fusion protein. Animals were killed by sodium pentobarbital
overdose at 72 hours postinjection, and various organs, blood, and
tumors were removed and weighed. The radioactivity in the samples
was then measured in a gamma counter. For each mouse, data were
expressed as percent injected dose/gram (% ID/g) and tumor:organ
ratio (cpm per gram tumor/cpm per gram organ). From these data,
the mean and SD were calculated for each group.
Fig 3. Competitive binding radioimmunoassay with chCLL-1/GMCSF and chCLL-1/IL-2. Purified antibody fusion proteins were assayed
for their ability to inhibit the binding of 125I-labeled muLym-2 to ARH77 human myeloma cells. muLym-2 and chLym-1 served as positive
and negative controls, respectively.
PCR. A PCR fragment containing either the human GMCSF cDNA or the human IL-2 cDNA preceded by a seven
amino acid linker peptide was then inserted into the Not I
site, producing CLL-1 VH/human g1/human GM-CSF or
CLL-1 VH/human g1/human IL-2 fusion genes (Fig 1). This
resulted in the expression vectors 12/chCLL-1/HL/GM-CSF
and 12/chCLL-1/HL/IL-2, encoding the chimeric light chain
and a fusion protein consisting of the chimeric CLL-1 heavy
chain with human GM-CSF or human IL-2 at its C-terminus.
The fusion proteins were expressed from NS0 murine myeloma cells using the glutamine synthetase gene amplifica-
Imaging Studies
ARH-77 human myeloma tumors were grown in the left thighs
of athymic nude mice as described above. When the tumors had
reached approximately 1 cm in diameter, the mice were injected IV
with a 0.1 mL inoculum containing 100 mCi/10 mg of 131I-labeled
chCLL-1, chCLL-1/GM-CSF, or chCLL-1/IL-2. At 1, 3, and 5 days
postinjection, the mice were anesthetized with a SC injection of 0.8
mg sodium pentobarbital. The immobilized mice were then imaged
in a prone position with a Spectrum 91 camera equipped with a
pinhole collimator (Raytheon Medical Systems, Melrose Park, IL)
set to record 5,000 to 10,000 counts using the Nuclear MAX Plus
image analysis software package (MEDX Inc, Wood Dale, IL).
RESULTS
Construction, Expression, and Purification of
chCLL-1/GM-CSF and chCLL-1/IL-2
A Not I site was previously appended immediately downstream of the terminal codon of the human g1 sequence by
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Fig 4. Colony-forming activity of chCLL-1/GM-CSF. Various concentrations of recombinant human GM-CSF, chCLL-1/GM-CSF, or
chCLL-1 were cultured with 7.5 Ì 104 bone marrow MNC in triplicate
in semisolid medium for 14 to 16 days at 377C until colonies containing more than 30 cells formed.
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GM-CSF AND IL-2 ANTIBODY FUSION PROTEINS
4441
centrations of chCLL-1/GM-CSF, chCLL-1/IL-2, muLym2, or an irrelevant MoAb (chLym-1) were evaluated for their
ability to inhibit the binding of 125I-labeled muLym-2 to
ARH-77 cells (Fig 3). Because it binds to a nonoverlapping
epitope, chLym-1 was unable to compete with 125I-labeled
Fig 5. Biologic activity of chCLL-1/IL-2 as determined by the ability
to support proliferation of CTLL-2 cells. Serial dilutions of chCLL-1/
IL-2, chCLL-1, or recombinant IL-2 standard were incubated with 2 Ì
104 CTLL-2 cells in triplicate for 20 hours at 377C. The cells were
pulsed with 0.5 mCi of 3H-thymidine for 6 hours, and the samples
were harvested and counted.
tion system (Celltech Biologics). After subjection to vector
amplification, the highest chCLL-1/GM-CSF–producing
subclone secreted approximately 26 mg/mL/106 cells/24
hours in static culture. The highest chCLL-1/IL-2–producing
subclone expressed approximately 16 mg/mL/106 cells/24
hours. Upon scale-up, greater than 100 mg/mL of chCLL-1/
GM-CSF were obtained after purification. When the chCLL1/IL-2–producing cell line was grown in a 10-L bioreactor,
approximately 70 mg/mL of fusion protein were obtained.
Both chimeric antibody fusion proteins were properly assembled as demonstrated by reducing SDS-PAGE; two welldefined bands were resolved for chCLL-1/GM-CSF at
approximately 25 and 66 kD and for chCLL-1/IL-2 at approximately 25 and 65 kD, corresponding to the molecular
weights of the immunoglobulin light chain and heavy chain
plus cytokine (Fig 2). Both fusion proteins appeared as a
single peak by HPLC analysis (data not shown).
Immunobiochemical Analysis
The immunoreactivity of purified chCLL-1/GM-CSF and
chCLL-1/IL-2 with the target antigen of muLym-2 was assessed by determining the binding to antigen-bearing ARH77 myeloma cells. In a radioimmunoassay, increasing con-
r
Fig 6. ADCC activity of chCLL-1 and fusion proteins. MoAb or
fusion protein (1 mg/mL) was cultured with ARH-77 human myeloma
target cells and human mononuclear effector cells at varying effector:target cell ratios as indicated. (A) Comparison between ADCC
mediated by muLym-2 and chCLL-1. (B) Comparison between ADCC
mediated by chCLL-1 and chCLL-1/IL-2. (C) Comparison between
ADCC mediated by chCLL-1 and chCLL-1/GM-CSF. Specific lysis with
the isotype-matched negative control (chTNT-1) was Ú5% (data not
shown). Expressed as mean Ô SD. At each effector:target cell ratio,
the difference between pairs is significant (P Ú .001).
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HORNICK ET AL
tion of the IL-2-dependent cell line compared with the recombinant IL-2 standard. This corresponds to a specific activity of approximately 8 1 105 IU/mg of fusion protein. At
higher concentrations (eg, ú1 nmol/L), maximum proliferation was achieved as evidenced by the plateau of the incorporation of 3H-thymidine into DNA. As expected, chCLL-1
had no activity.
Cytotoxicity Studies
Fig 7. Whole body pharmacokinetic clearance of 125I-labeled
chCLL-1, chCLL-1/GM-CSF, and chCLL-1/IL-2 in nontumor-bearing
mice. Activity at injection and at selected times thereafter was measured with a microdosimeter.
muLym-2, but chCLL-1/GM-CSF and chCLL-1/IL-2 inhibited 125I-labeled muLym-2 binding to ARH-77 cells. These
studies confirm that chCLL-1/GM-CSF and chCLL-1/IL-2
maintain the immunoreactivity of muLym-2.
Avidity binding studies were then conducted in which 125Ilabeled chCLL-1/GM-CSF or chCLL-1/IL-2 was incubated
with ARH-77 cells and the bound radioactivity used to calculate the avidity constant Ka by Scatchard analysis as described in the Materials and Methods. chCLL-1/GM-CSF
and chCLL-1/IL-2 had similar binding constants of 3.3 1 108
mol/L01 and 3.0 1 108 mol/L01, respectively. The binding
constant of muLym-2 was determined to be 2.9 1 108 mol/
L01. These studies demonstrate that the presence of the cytokines on the C-terminus of the heavy chain does not affect
binding to the antigenic target.
Colony-Forming Activity of chCLL-1/GM-CSF
Biologic activity of the GM-CSF moiety was determined
by colony assays using both bone marrow MNC and CD34/
cells. As indicated in Fig 4, chCLL-1/GM-CSF compares
favorably with recombinant human GM-CSF in its ability to
stimulate colony formation from the MNC fraction of normal
bone marrow. In addition, the fusion protein is capable of
inducing the formation of colonies from isolated CD34/
progenitor cells (data not shown). No colonies formed in the
presence of chCLL-1.
IL-2 Bioactivity of chCLL-1/IL-2
Biologic activity of the IL-2 moiety was determined by
assaying the ability of chCLL-1/IL-2 to support IL-2-dependent T-cell proliferation. A bioassay with the IL-2–dependent CTLL-2 line was performed in which chCLL-1/IL-2
was assayed along with chCLL-1 and the IL-2 standard (Fig
5). On a molar basis, chCLL-1/IL-2 had approximately 50%
of the activity required to produce 50% maximum prolifera-
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chCLL-1/GM-CSF, chCLL-1/IL-2, chCLL-1, and muLym2 were evaluated for their ability to mediate ADCC by colorimetric lactate dehydrogenase release assays against ARH77 myeloma target cells. At a concentration of 1 mg/mL,
chCLL-1 mediated 65% cytotoxicity, while muLym-2 mediated only 10% specific lysis of tumor cells by human MNC
at an effector:target cell ratio of 50:1 (Fig 6A). At the same
effector:target cell ratio, both fusion proteins mediated approximately 100% specific lysis of target cells (Fig 6B and
C). Similar enhancement of specific lysis mediated by both
fusion proteins over chCLL-1 and by chCLL-1 over muLym2 can be seen at lower effector:target cell ratios. The isotypematched irrelevant control (chTNT-1) mediated õ5% specific lysis at all effector:target cell ratios (data not shown).
Neither the fusion proteins nor antibodies mediated specific
lysis of target cells by human PMN at an effector:target cell
ratio of 50:1 (õ5%, data not shown).
In Vivo Pharmacokinetic and Tumor Targeting Studies
Whole body clearance studies were performed to establish
differences in pharmacokinetics among chCLL-1/GM-CSF,
chCLL-1/IL-2, and chCLL-1. Mice were injected with 125Ilabeled fusion proteins or chimeric antibody, and the whole
body activity at injection and selected times thereafter was
measured with a microdosimeter. chCLL-1/IL-2 cleared rapidly with a whole body half-life of 11 hours (Fig 7). chCLL1/GM-CSF had a half-life of approximately 30 hours, while
chCLL-1 cleared slowly, with a half-life of 100 hours.
The difference among clearance rates was evident when
tumor and normal organ biodistribution was examined in
ARH-77 myeloma-bearing nude mice. As indicated in Fig
8A, tumor uptake of chCLL-1 after 72 hours was 2.54% {
0.14% injected dose/gram, while tumor uptake of chCLL-1/
IL-2 and chCLL-1/GM-CSF was significantly lower (1.14
{ 0.08 and 1.07 { 0.10, respectively; P õ .001). However,
uptake of the fusion proteins in normal tissues was considerably lower than chCLL-1, which can be attributed to the
rapid clearance of the fusion proteins. This low normal tissue
uptake produces higher tumor/organ ratios, as can be seen
in Fig 8B.
Imaging studies were also performed to examine tumor
targeting with the fusion proteins. Tumor-bearing nude mice
were injected with 131I-labeled chimeric antibody or fusion
protein and imaged at 1, 3, and 5 days postinjection. In Fig 9,
the difference in clearance is manifested by the unambiguous
localization of the fusion proteins to the tumor after 24 hours,
while the mouse injected with chCLL-1 demonstrated high
signal throughout the body at 1 and 3 days postinjection.
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GM-CSF AND IL-2 ANTIBODY FUSION PROTEINS
4443
Fig 8. Tissue biodistribution and tumor uptake
of chCLL-1, chCLL-1/GM-CSF, and chCLL-1/IL-2 at 72
hours postinjection in ARH-77 myeloma tumor-bearing nude mice. (A) Tumor uptake measured by percent injected dose/gram of 125I-labeled MoAb or fusion protein in the indicated tissues. (B) Tumor:organ
ratios expressed as mean Ô SD.
Nevertheless, by day 5, localization of chCLL-1 to the tumor
site is clear. In mice that received chCLL-1/GM-CSF or
chCLL-1/IL-2, by day 5 no signal remained except in the
tumor. These data demonstrate that chCLL-1/GM-CSF and
chCLL-1/IL-2 effectively localize to the ARH-77 human
myeloma xenografts.
DISCUSSION
In this study, recombinant fusion proteins containing the
chimeric MoAb CLL-1 and human GM-CSF or IL-2 have
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been generated, which retain both tumor targeting and cytokine functions. The GS gene amplification system was used
for high level expression of the fusion proteins from myeloma cells so that large-scale production can yield sufficient
products to enable clinical studies to be undertaken. With
this expression system, gram quantities of the fusion proteins
can be produced in batch cultures. Biochemical analysis
demonstrates the presence of two GM-CSF or IL-2 molecules per chimeric antibody molecule (Fig 2). GM-CSF or
IL-2 is located at the C-terminus of the heavy chain follow-
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4444
HORNICK ET AL
Fig 9. Imaging of ARH-77 myeloma tumor-bearing nude mice
injected with 131I-labeled chCLL1 (A), chCLL-1/GM-CSF (B), or
chCLL-1/IL-2 (C). Mice were imaged in a prone position at the
indicated times postinjection.
ing a short linker peptide to facilitate proper folding of the
cytokine. The immunoreactivity of the fusion proteins was
retained, as evidenced by competition with 125I-labeled
muLym-2 for binding to antigen-bearing ARH-77 myeloma cells (Fig 3). Moreover, the binding affinity of the
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fusion proteins was unaffected by the presence of the cytokine molecules. In addition, the biological activity of the
cytokines within the fusion proteins was confirmed by
appropriate assays; chCLL-1/GM-CSF possesses colonyforming activity (Fig 4), while chCLL-1/IL-2 is able to
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GM-CSF AND IL-2 ANTIBODY FUSION PROTEINS
4445
support the proliferation of an IL-2 – dependent T-cell line
(Fig 5).
Cytotoxicity studies clearly demonstrate the improved effector functions of chCLL-1 over muLym-2 and of both
fusion proteins over chCLL-1 (Fig 6). Human IgG1 constant
regions were selected for construction of the chimeric MoAb
based on earlier observations of the enhanced antitumor cytotoxic activity of chimeric IgG1 over chimeric MoAbs of
other isotypes.33 At each effector:target cell ratio, chCLL-1/
IL-2 mediates higher specific tumor lysis by human MNC
than the chimeric MoAb alone (Fig 6B). This is in agreement
with previous reports of augmented MNC ADCC either by
free recombinant IL-24-7,9 or by a recombinant MoAb/IL-2
fusion protein.34,35 chCLL-1/GM-CSF also mediates higher
specific tumor lysis by human MNC than chCLL-1 (Fig 6C).
Ragnhammar et al36 have previously shown that short-term
preincubation of MNC with GM-CSF enhances ADCC
against colorectal carcinoma and lymphoma cell lines. Other
investigators have observed no effect of GM-CSF on MNC
ADCC against malignant B-cell lines.9 There is evidence
that GM-CSF and IL-2 can act synergistically in vitro. In this
regard, GM-CSF has been shown to augment the induction of
lymphokine-activated killer (LAK) activity by IL-2 against
a human Burkitt’s lymphoma cell line through monocytes.37
In addition, GM-CSF and IL-2 enhance ADCC against a
colorectal carcinoma cell line,38 leading the investigators to
suggest combination therapy consisting of low dose IL-2,
GM-CSF, and MoAb.
No specific lysis of target cells by PMN in ADCC mediated by chCLL-1 or chCLL-1/GM-CSF was observed in our
studies. It has recently been demonstrated that antibodies
recognizing HLA class II mediate lysis of malignant B-cell
lines by PMN, while antibodies to other B-cell antigens fail
to mediate such ADCC.32 In these studies, Lym-1 and 1D10,
both of which recognize HLA class II related epitopes, did
not mediate ADCC by PMN from healthy donors, although
both MoAbs mediated ADCC with PMN from patients
treated with granulocyte colony-stimulating factor. Similar
results have been observed with Lym-1 in combination with
GM-CSF.9 GM-CSF has also been shown to enhance PMN
ADCC against solid tumor cell line targets, including neuroblastoma, melanoma, and colorectal carcinoma.36,39,40 It is as
yet unclear why MoAbs directed against particular antigens
on malignant B cells possess the ability to mediate PMN
ADCC, while those with specificity for other B-cell antigens
do not. Based on in vitro ADCC data and clinical experience
with a murine MoAb, a clinical trial using the combination
of the MoAb and GM-CSF for the treatment of metastatic
colorectal carcinoma was initiated.41 In this study, complete
remissions were achieved in some patients, providing clinical evidence for the benefit of combination therapy.
In the current study, pharmacokinetic analysis in Balb/C
mice demonstrated the marked difference in whole body
clearance among chCLL-1 and the fusion proteins (Fig 7).
We have recently shown that a fusion protein consisting of
chLym-1 and IL-2 has a half-life of 11 hours,35 which is
identical to that observed for chCLL-1/IL-2. chCLL-1/GMCSF has a whole body half-life intermediate between the
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chimeric MoAb and the IL-2–containing fusion protein. The
relatively longer half-life of a GM-CSF–containing antibody
fusion protein compared with those containing other cytokines has previously been described for the antiganglioside
MoAb ch14.18.42 It has yet to be demonstrated whether similar differences in clearance between chimeric MoAbs and
cytokine-containing antibody fusion proteins exist in patients. Biodistribution and imaging studies in human myeloma-bearing nude mice illustrate the tumor targeting abilities of chCLL-1/IL-2 and chCLL-1/GM-CSF (Figs 8 and 9).
Despite their rapid clearance profiles, they retain the capacity
to localize to tumor xenografts effectively. In fact, such rapid
clearance might be beneficial in clinical applications,
wherein potentially injurious cytokine exposure to normal
tissues would be minimized. This is particularly true for IL2, which induces a capillary leak syndrome when administered systemically in high doses.43-46
There is considerable evidence that high local concentrations of cytokines within tumors can stimulate antitumor
immunity and rejection in animal models. The majority of
such efforts have employed gene transfection to engineer
tumor cell lines to secrete cytokines.10-17 Although these studies demonstrate the utility of delivering cytokines directly
to tumors, they are presently impractical in the clinical setting. A more feasible approach to generating high local concentrations of cytokines within tumors is targeting cytokines
via antibody fusion proteins.18,19 This approach combines the
cytotoxicity that MoAbs can mediate against tumor targets
with the host antitumor immune response, which is stimulated by high local concentrations of cytokines. Several
groups have taken such an approach to delivering cytokines
by engineering fusion proteins consisting of IL-2 and antibody fragments including F(ab*)19 and single-chain antibodies.47-49 Intact MoAbs may have greater effectiveness than
fragments, however, because they can mediate ADCC. An
alternative approach that also employs antibody-cytokine fusion proteins is engineering a cancer vaccine using idiotypecytokine fusion proteins including IL-2 and GM-CSF.50,51 In
a murine B-cell lymphoma model, such fusion proteins have
been shown to induce antitumor responses. The efficacy of
antibody-targeted IL-2 has been elegantly demonstrated in
both a SCID mouse human neuroblastoma model20,52 and a
syngeneic murine melanoma model.53,54 In these studies, the
effector cell population responsible for antitumor responses
was identified as CD8/ T cells. As the fusion protein retained
a therapeutic effect in natural killer (NK) cell-deficient mice,
the investigators concluded that tumor eradication was not
dependent on NK cells.55 Whether such a mechanism of
antitumor cytotoxicity holds for other antibody-cytokine fusion proteins in the treatment of other malignancies remains
to be determined.
The chimeric antibody fusion proteins described in the
current study have the potential for producing tumor killing
by a number of mechanisms. The parent muLym-2 is reactive
with a majority of human B-cell lymphomas, chronic
lymphocytic leukemias, and multiple myeloma,21 suggesting
that this MoAb and derivatives may be of use in treating a
variety of B-cell malignancies. Both muLym-2 and chCLL-
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4446
HORNICK ET AL
1 have a direct inhibitory effect on human lymphoma cell
lines and can improve the survival of SCID mice injected
with human lymphoma cells (Funakoshi et al, in preparation). Furthermore, both chCLL-1/GM-CSF and chCLL-1/
IL-2 mediate enhanced ADCC against a human myeloma
cell line. Finally, the combination of GM-CSF and IL-2
targeted to the tumor site may be sufficient to bring about
the induction of effective cytotoxic T-cell responses. As the
antigen recognized by chCLL-1 is not present in animal
lymphomas and hence a syngeneic model is unavailable in
which to evaluate immune responses induced by chCLL-1/
GM-CSF and chCLL-1/IL-2, clinical trials will be undertaken to test the immunotherapeutic efficacy of these novel
reagents against human B-cell malignancies.
ACKNOWLEDGMENT
The authors wish to thank Barbara H. Biela, Jahangir Sharifi, and
Myra M. Mizokami for assistance with the animal studies.
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1997 89: 4437-4447
Chimeric CLL-1 Antibody Fusion Proteins Containing
Granulocyte-Macrophage Colony-Stimulating Factor or Interleukin-2 With
Specificity for B-Cell Malignancies Exhibit Enhanced Effector Functions
While Retaining Tumor Targeting Properties
Jason L. Hornick, Leslie A. Khawli, Peisheng Hu, Maureen Lynch, Peter M. Anderson and Alan L. Epstein
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