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Department of Microbiology, Immunology, and Molecular Genetics and the Molecular Biology Institute, University of California, Los Angeles, CA 90095 L.S.P. is a student in the University of California, Los Angeles Medical Scientist Training Program and is supported by grant GM08042 and the Aesculapians Fund of the University of California, Los Angeles School of Medicine.
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Two approaches to achieving high levels of cytokine at the site of the tumor have been direct injection of cytokine into the tumor or transfer of the gene encoding the cytokine into tumor cells (7). While both methods have been shown to be effective, they also have significant limitations: direct injection into micrometastases is not possible, and currently gene transfer involves ex vivo manipulation of tumor cells, which makes treatment of large numbers of patients difficult and costly. Abs provide an alternative specific delivery vehicle in which tumor-specific Abs can be used to selectively target a metastatic/residual nodule and deliver an immunostimulatory molecule like a cytokine. The specific targeting should make it possible to elicit a systemic tumor-specific immune response without accompanying systemic toxicity.
There are many different types of tumor-associated Ags: oncofetal Ags (e.g., carcinoembryonic Ag (8)), Ags expressed on cells at a particular stage of differentiation (e.g., IL-2R (9)), growth factor receptors (e.g., transferrin receptor (10)), oncogene products (e.g., c-myc (11)), and the Id expressed by the surface Ig of lymphoma cells (12). These tumor-associated Ags distinguish normal from tumor tissue and have been used as targets for cancer therapy (13, 14, 15, 16, 17). Her2/neu, also known as c-erbB-2, is a cell surface oncogene product that is amplified and/or overexpressed in 25–30% of human breast and ovarian cancers with this overexpression associated with poor prognosis (18, 19). Humanized anti-Her2/neu has been shown to be an effective therapeutic agent in clinical trials (20). These trials demonstrate that metastatic breast cancer can be effectively targeted through the Her2/neu Ag and suggest that Abs specific for Her2/neu would be effective vehicles for targeting cytokines to the sites of the tumors.
Several different cytokines are attractive candidates for enhancing tumor-specific immune responses. IL-2 induces the proliferation of T cells, supports the growth of Ag-specific T cell clones, and enhances the activity of T and NK cells (21). Fusion of IL-2 to Abs specific for tumor-associated Ags such as ganglioside GD2 and the Id of a murine lymphoma has resulted in fusion proteins that have shown much promise as agents for stimulating tumor-specific immune responses (22, 23, 24, 25, 26, 27). Indeed, an IL-2-Ab fusion protein specific for GD2 was able to generate an immune response that eliminated metastatic disease in a murine model of melanoma (22).
Another cytokine that has great potential for use in tumor
immunotherapy is IL-12. IL-12 is a heterodimeric cytokine with many
actions on innate and cellular immunity that may have antitumor and
antimetastatic effects. IL-12 can activate T and NK cells, induce the
production of IFN-
, and stimulate naive CD4+ T
cells to differentiate toward the Th1 phenotype (28, 29). A Th1
response involves the secretion of a cytokine profile that activates
cytotoxic T cells and macrophages, which could be desirable in an
antitumor immune response. In addition, IL-12 may act through
nitric oxide to cause cell-cycle arrest of tumor cells (4), and
through induction of inducible protein-10 to inhibit angiogenesis
(30).
Bioactive IL-12 requires the expression of two separate genes, p40 and p35, and correct heterodimer assembly (31). To address this issue, Gillies et al. have recently reported the construction of an Ab-IL-12 fusion protein in which the p35 subunit was fused to the carboxyl terminus of an Ab; the p40 subunit was expressed as a separate polypeptide that must then assemble with the p35 subunit. Although this IL-12/Ab fusion protein was functional, the IL-12 bioactivity was 2-fold lower than rIL-12 (32). An alternative that eliminates the need to assemble two independently produced peptides is to express IL-12 as a single chain with the p40 and p35 subunits joined by a flexible linker. We have now used this alternative approach and constructed an Ab fusion protein in which murine single-chain (msc)3 IL-12 (p40.linker.p35) is fused to an anti-Her2/neu Ab at the amino terminus of the H chain (mscIL-12.her2.IgG3). Importantly, this fusion protein retains Ab specificity, exhibits IL-12 bioactivity comparable to recombinant murine (m) IL-12, and demonstrates antitumor activity in vivo.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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P3X63Ag8.653 cells (American Type Culture Collection, Manassas, VA), CT26 cells (murine colon adenocarcinoma cells kindly provided by Young Chul Sung, Pohang University, Korea), and CT26/Her2 cells (developed in our laboratory by transfection of CT26 cells with the cDNA encoding Her2/neu using methods previously described (33)) were cultured in IMDM supplemented with 5% bovine calf serum, L-glutamine, penicillin, and streptomycin. K562 cells (American Type Culture Collection) were cultured in RPMI 1640 supplemented with 10% FBS, sodium pyruvate, HEPES, and D-glucose. Kit255/K6 cells (kindly provided by Jim Johnston, DNAX, Palo Alto, CA) were maintained in RPMI 1640 supplemented with 10% FBS and 100 IU recombinant human (h) IL-2/ml (kindly provided by Chiron, Emeryville, CA). rmIL-12 reference standard was kindly provided by Stanley Wolf (Genetics Institute, Cambridge, MA).
Mice
Female 6- to 8-wk-old BALB/c mice were obtained from Taconic Farms (Germantown, NY) and conventionally housed. All experiments were performed according to the National Institutes of Health Guide for the Care and Use of Laboratory Animals.
Ab expression vectors
her2.IgG3.
The variable L and H chain domains were obtained from the plasmid pAK19
containing the humanized humAb4D5–8 Ab (generously provided by Paul
Carter, Genentech, South San Francisco, CA) (34, 35) and cloned as
previously described (36) into mammalian expression vectors for human
L chain and IgG3 H chain, respectively.
mscIL-12.her2.IgG3.
The cDNA for mscIL-12 (p40 subunit linked by a
(Gly4Ser)3 flexible linker
to the p35 subunit from which the first 22 aa (leader sequence) were
deleted) was generously provided by Richard Mulligan (Harvard Medical
School, Boston, MA) as plasmid pSP72.mIL-12.p40.linker.
p35. mscIL-12
was amplified from the plasmid by PCR using the sense primer
5'-CCCCAAGCTTGATATCCACCATGGGTCCTCAGAAGCTAACC-3' and the antisense
primer 5'-CCCGAATTCGTTAACCGGCGGAGCTCAGATAGCCC-3'. The PCR product
was cloned as a HindIII/HpaI fragment to the 5'
end of a cassette encoding the
(Gly4Ser)3 linker sequence
of Huston et al. (37) fused to the anti-Her2/neu
VH sequence. The resulting
mscIL-12.linker.VH coding sequences were excised
as an EcoRV/NheI fragment and cloned into an
expression vector for human IgG3 H chain (38).
Recombinant Ab expression, immune precipitation, and purification
Transfection, expression, and purification of the recombinant
Abs were performed as previously described (39) to obtain
mscIL-12.her2.IgG3. Briefly, 1 x 107
P3X63Ag8.653 myeloma cells were transfected by electroporation with 10
µg of each of the mscIL-12.her2.IgG3 H and anti-Her2 L chain
expression vectors (linearized with PvuI). Transfected cells
were plated at 2 x 104 cells/well in a
flat-bottom 96-well tissue culture plate and selected with the addition
of 10 mM histidinol (Sigma, St. Louis, MO) on days 3 and 5 after
transfection. Wells were screened for Ab secretion after 10–14 days by
ELISA using 96-well flat-bottom plates coated with goat anti-human
IgG (Zymed, South San Francisco, CA). Supernatant from the transfected
cells was applied, followed by the addition of goat anti-human
conjugated with alkaline phosphatase (Sigma). Binding was detected by
the addition of phosphatase substrate
(para-nitrophenyl phosphate, disodium; Sigma), and
positive wells were expanded.
To determine the size and assembly pattern of the secreted recombinant
mscIL-12.her2.IgG3 Ab, supernatants from cells grown overnight in
medium containing [35S]methionine (Amersham,
Piscataway, NJ) were immunoprecipitated with polyclonal rabbit
anti-human IgG and rabbit anti-human (produced by Letitia
A. Wims in our laboratory), followed by staphylococcal protein A
(IgGSorb; The Enzyme Center, Malden, MA). Precipitated Abs were
analyzed on SDS-polyacrylamide gels in the presence or absence of
reducing agent (2-ME).
For the purification of mscIL-12.her2.IgG3, high producing clones were
expanded in roller bottles in IMDM plus 1% fetal clone serum plus
Glutamax (Life Technologies, Rockville, MD), and cell-free culture
supernatant was collected. Culture supernatants were passed through a
protein A column, the column was washed with 10 ml PBS, and the
proteins were successively eluted with 2 ml of 1 M citric acid, pH 4.5,
5 ml of 0.1 M glycine, pH 2.5, and 2 ml of 0.1 M glycine, pH 2.0. The
eluted fractions were neutralized immediately with 2 M Tris-HCl, pH
8.0. The fractions were concentrated using Ultra-free-15 filters
(Millipore, Bedford, MA) with a cut-off of 100 kDa and dialyzed. Using
this method, 2 L of culture supernatant yields 
0.8 mg
mscIL-12.her2.IgG3.
Assays of binding to Her2/neu Ag and IL-12R
Ag binding. CT26 or CT26/Her 2 were incubated with mscIL-12.her2.IgG3, her2.IgG3, or dansyl.IgG3 (an IgG3 isotype control Ab specific for the hapten dansyl) for 1 h at 4°C. The cells were washed and incubated 2 h at 4°C with PE-labeled goat anti-human IgG (PharMingen, San Diego, CA) and analyzed by flow cytometry. Analysis was performed with a FACScan (Becton Dickinson, Mountain View, CA) equipped with a blue laser excitation of 15 mW at 488 nm.
Persistence of Ab binding at the cell surface. CT26/Her2 cells were incubated with mscIL-12.her2.IgG3, her2.IgG3, or dansyl.IgG3. The cells were washed and incubated at 37°C in culture medium. At different time points (0, 1, 4, and 24 h), an aliquot of cells was removed and stained with PE-conjugated anti-human IgG for FACS analysis. The mean fluorescence was calculated as a percentage of the maximum mean fluorescence at time zero.
Binding to IL-12R. Kit225/K6 cells, a subclone of the human T leukemic cell line that expresses the IL-12R (40), were incubated with her2.IgG3 or mscIL-12.her2.IgG3. Binding was assayed by staining with PE-conjugated anti-human IgG followed by FACS analysis. In a second assay, PHA-activated PBMC were incubated with her2.IgG3 or mscIL-12.her2.IgG3. PBMC have been shown to express IL-12Rs following activation with PHA and IL-2 (41). Binding was assayed by staining with PE-conjugated anti-human IgG followed by FACS analysis.
Proliferation assays
Proliferation assays were performed as previously described (42). PBMC were isolated from normal blood donors by Ficoll-Hypaque density centrifugation (Ficoll-Paque, Pharmacia, Piscataway, NJ). These cells were then depleted of monocytes by plastic adherence, and nonadherent cells were resuspended at 5 x 105 cells/ml in supplemented medium [1:1 complete RPMI 1640:complete DMEM plus 5% human AB serum (Irvine Scientific, Santa Ana, CA), 10 mM HEPES, 0.006% (w/v) L-arginine monohydride, and 0.1% (w/v) dextrose] containing 2 µg/ml PHA-P (Difco Laboratories, Detroit, MI) and were cultured for 3 days. Cells were then split 1:1 with fresh supplemented medium containing 20 IU/ml rhIL-2 (kindly provided by Chiron Corporation) and incubated for a further 24–48 h. The PHA blasts were then washed with acidified RPMI 1640, pH 6.4, and rested in RPMI 1640 plus 0.5% human AB serum for 3–4 h. The cell concentration was adjusted to 2 x 106 cells/ml in supplemented media. Neutralizing anti-IL-2 Ab (BioSource International, Camarillo, CA) was added at 1 µg/ml to block IL-2-induced proliferation.
Serial 1:3 dilutions of equivalent protein concentrations of mIL-12, mscIL-12.her2.IgG3, and her2.IgG3 were made in supplemented medium over a range of 36 ng/ml to 16 pg/ml. Next, 50 µL cell suspension was mixed with 50 µL mIL-12, mscIL-12.her2.IgG3, her2.IgG3, or supplemented medium in triplicate in a flat-bottom 96-well tissue culture plate. After 48 h of culture at 37°C, 5% CO2, proliferation was measured by the 3-(4, 5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)- 2-(4-sulfophenyl)-2H-tetrazolium, inner salt (MTS)/phenazine methosulfate (PMS) assay (Promega, Madison, WI), and plates were read at OD490.
Enhanced NK activity of PBMC
These assays were performed according to the methods of Hatam et al. (43) with modifications. Briefly, effector PBMC were isolated as described above, then resuspended in RPMI 1640 plus 10% FBS at 1–2 x 106 cells/ml. rmIL-12 at 5 ng/ml, an equivalent IL-12 concentration of mscIL-12.her2.IgG3, or an equivalent Ab concentration of her2.IgG3 were added to the PBMC and incubated for 16–18 h at 37°C, 5% CO2. The cell concentration was then adjusted to 0.5–1 x 106 cells/ml. Target K562 cells (2 x 107) were washed two times with serum-free RPMI 1640, then resuspended in 1 ml Diluent C (Sigma). Then, 4 µM PKH67 was prepared by diluting the stock solution (Sigma) in Diluent C. The cell suspension and dye were mixed in equal volumes (1 ml each) in a polypropylene tube and incubated at room temperature for 2 min. An equal volume (2 ml) of FBS was added to stop the labeling reaction. The cells were washed three times with RPMI 1640 plus 10% FBS and resuspended at 1–2 x 105 cells/ml in RPMI 1640 plus 10% FBS.
For the NK cytotoxicity assay, 100 µL effector PBMC and 100 µL PKH67-labeled K562 cells were added to polystyrene 12 x 75 mm tubes to create E:T ratios of 50:1 and 100:1 and were incubated for 4 h at 37°C, 5% CO2. At the end of the incubation, 0.5 ml isotonic propidium iodide (PI) at 5 µg/ml (Sigma) was added to each tube and immediately analyzed by FACS. Spontaneous cell death was determined by incubating either targets or effectors alone.
FACS analysis was performed with a FACScan (Becton Dickinson) equipped with a blue laser excitation of 15 mW at 488 nm. The two fluorochromes, PKH67 and PI, were electronically compensated using PKH67-labeled targets alone and unstained target cells whose membrane had been permeabilized by treatment with 0.1% Tween-20 in PBS for 10 min at 37°C. These cells were then washed twice and 0.5 ml of isotonic PI added before FACS analysis. Data were collected in list mode and analyzed using Cell Quest software (Becton Dickinson). At least 2000 target events were collected per sample. Percent cytotoxicity was calculated as (number of dead targets)/(total number of targets) x 100.
In vivo antitumor activity
A total of 1 x 106 CT26/Her2 cells in 0.15 ml PBS were injected s.c. into the right flank of syngeneic BALB/c mice on day 0. One group of mice was treated i.v. with mscIL-12.her2.IgG3 (at a concentration equivalent to 1 µg IL-12/day), her2.IgG3 (at a concentration equivalent to the Ab concentration of mscIL-12.her2.IgG3 administered/day), or PBS for 5 days beginning on day 1. A second group of mice was similarly treated beginning on day 6. In each group, 10 mice per treatment arm were used. Tumor growth was monitored and measured with a caliper every other day beginning on day 6 and continuing until day 20. At that point, all mice were euthanized and the tumors were harvested and weighed.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The construction of her2.IgG3 and vectors for the production of H
chain fusion proteins was previously described (36). For the present
studies, we elected to use mIL-12 in our fusion protein because mIL-12
is biologically active on activated murine and human T and NK cells,
while murine T and NK cells do not respond to hIL-12 (44). mscIL-12 was
amplified from plasmid pSP72.mIL-12.p40.linker.p35 and cloned at the
amino terminus of the VH region of her2.IgG3
(Fig. 1
). A flexible
(Gly4Ser)3 linker was
positioned between IL-12 and the V region to facilitate both correct
folding of the Ab and IL-12 and simultaneous Ag and IL-12R binding. The
mscIL-12.her2.IgG3 H chain and L chain were cotransfected into
P3X63Ag8.653 myeloma cells, and stable transfectants secreting Ab were
selected using the anti-human IgG ELISA described in
Materials and Methods.
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followed by insoluble protein A
were added to the supernatant and cell lysates, and the
immunoprecipitates were analyzed by SDS-PAGE. In the absence of
reducing agents, IgG3 migrates with an apparent molecular mass of 170
kDa, while mscIL-12.her2.IgG3 is about 320 kDa, the expected molecular
mass of the fusion protein (Fig. 2A). Following treatment with
the reducing agent 2-ME, L chains migrating with an apparent molecular
mass of 25 kDa are seen for both proteins. However, the IgG3 has a H
chain of 60 kDa, while mscIL-12.her2.IgG3 has a H chain of 135
kDa. Thus, proteins of the expected molecular mass are produced, and
fusion of scIL-12 to her2.IgG3 does not appear to alter the assembly
and secretion of the H2L2
form of the Ab.
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The ability of mscIL-12.her2.IgG3 to bind to the
Her2/neu antigenic target was examined using flow cytometry.
Both mscIL-12.her2.IgG3 (Fig. 3B) and her2.IgG3 (Fig. 3C) specifically bound to CT26/Her2; neither Ab bound to
parental CT26 cells (data not shown). Importantly, the same
fluorescence intensity was seen with both her2.IgG3 and
mscIL-12.her2.IgG3, suggesting that both have similar affinity for
Her2/neu. A control IgG3 Ab specific for the hapten dansyl
did not bind to CT26/Her2 (Fig. 3A). These data indicate
that the fusion of a 75-kDa scIL-12 to the amino terminus of each H
chain of her2.IgG3 does not interfere with the ability of the Ab to
recognize the Her2/neu Ag.
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).
These results indicate that fusion of the Ab with IL-12 does not affect
the dissociation rate, internalization, or degradation of
mscIL-12.her2.IgG3 at the surface of the cell compared with her2.IgG3.
This suggests that the IL-12 in our fusion protein will be present at
the cell surface to activate T and NK cells.
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The ability of mscIL-12.her2.IgG3 to bind to the IL-12R was
determined by flow cytometry of both transformed and normal human
cells. The mscIL-12.her2.IgG3 bound to Kit225/K6, a subclone of the
human T leukemic cell line that expresses the IL-12R (40), while
her2.IgG3 did not (Fig. 5A).
Neither her2.IgG3 or mscIL-12.her2.IgG3 bound to the resting PBMC (Fig. 5B). The mscIL-12.her2.IgG3 bound to the PHA-activated PBMC
while her2.IgG3 did not (Fig. 5C). These results show that
the IL-12 in the fusion protein is able to bind to the IL-12R.
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After establishing that mscIL-12.her2.IgG3 was correctly assembled, secreted, and retained the ability to bind both the Her2/neu Ag and the IL-12R, we investigated its biologic activity. All assays of IL-12 biological activity were expressed relative to the IL-12 concentration used (i.e., ng/ml). To obtain the IL-12 concentration of the fusion protein, the fraction of the IL-12-Ab fusion protein that was IL-12 (150 kDa/320 kDa) was multiplied times the protein concentration of the fusion protein. In this way, the biological activity of rIL-12 and IL-12 in the fusion protein could be compared on a per molecule basis. Similarly, to ensure that equivalent Ab concentrations of her2.IgG3 and mscIL-12.her2.IgG3 were used, the fraction of the Ab-fusion protein that was Ab (170 kDa/320 kDa) was multiplied times the protein concentration of the fusion protein to obtain the Ab concentration of the fusion protein. The same concentration of her2.IgG3 was then used as a control.
One of the pleiotropic actions of IL-12 is the ability to induce the
proliferation of PHA-activated lymphoblasts. We prepared PHA-activated
PBMC and incubated them for 48 h with mIL-12, mscIL-12.her2.IgG3,
or her2.IgG3. Proliferation was measured by addition of MTS/PMS. Fig. 6 shows the results from a typical assay.
mIL-12 and mscIL-12.her2.IgG3 showed an equivalent mitogenic effect on
PHA-blasts in a dose-dependent manner. The results are expressed as the
mean ± SD of triplicate samples with the background proliferation
in medium subtracted. In contrast, her2.IgG3-treated PHA-blasts did not
show any proliferation. These results indicate that the mitogenic
effect of mscIL-12.her2.IgG3 is due to the IL-12 and not to some other
effect by the Ab component of the fusion protein.
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IL-12 has been shown to enhance the cytotoxic action of NK cells.
We prepared PBMC (shown by FACS to be 8–9%
CD56+, data not shown) and incubated them for
16–18 h with 5 ng/ml mIL-12 or an equivalent IL-12 concentration of
mscIL-12.her2.IgG3. The PBMC were also incubated with her2.IgG3 at the
same Ab concentration as mscIL-12.her2.IgG3 or medium. These effector
cells were added to PKH67-labeled K562 target cells at E:T ratios of
100:1 and 50:1, then incubated for 4 h. After this incubation, PI,
which intercalates into the DNA of dead cells, was added and FACS
analysis performed. Fig. 7A
shows a representative FACS result (E:T of 100:1, treated with 5 ng/ml
mIL-12) with defined populations of live effectors (lower
left), dead effectors (upper left), live targets
(lower right), and dead targets (upper
right) separated into the four quadrants. The x-axis
measures PKH67 fluorescence intensity and the y-axis
measures PI fluorescence intensity. A gate was drawn around target
cells (PKH67 positive). Figs. 7, B–E show histograms of PI
fluorescence intensity among target cells gated as in Fig. 7A. More positively staining cells (right) are
dead target cells; less positive cells (left) are
live target cells. The percent cytotoxicity was calculated from the
histograms. The background cytotoxicity in medium was subtracted to
give the percent enhanced cytotoxicity. Fig. 7E shows that
mIL-12 and mscIL-12.her2.IgG3 at equivalent IL-12 concentrations of 5
ng/ml and E:T of both 50:1 and 100:1 comparably enhanced NK
cytotoxicity by 20%, while her2.IgG3 showed no enhancement. These
results indicate that the enhanced cytotoxicity by mscIL-12.her2.IgG3
is due to the IL-12 component of the fusion protein and not to some
other effect by the Ab component of the fusion protein and that the
IL-12 in the fusion protein has activity comparable to rIL-12.
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After demonstrating that mscIL-12.her2.IgG3 had in vitro biologic activity comparable to rIL-12, the in vivo antitumor activity was investigated using a CT26/Her2 animal model developed in our laboratory (33).
On day 0, CT26/Her2 cells were injected s.c. into the right flank of
BALB/c mice. One group of mice was treated with mscIL-12.her2.IgG3,
her2.IgG3, or PBS injected i.v. for 5 days beginning on day 1, while a
second group was similarly treated beginning on day 6 when the
tumors averaged 8–9 mm in diameter. Thus, the studies were designed to
examine the effect of mscIL-12.her2.IgG3 on both tumor growth and tumor
regression. Treatment with mscIL-12.her2.IgG3 slowed the growth of
tumors when it began on day 1 (Fig. 8A) and arrested tumor growth
when it began on day 6 (Fig. 8C) compared with mice treated
with PBS or her2.IgG3. The tumor weights were used as a more objective
indicator of tumor size and confirm the results of the caliper
measurements (Fig. 8, B and D).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Previous studies suggested that an accessible N terminus of the p40 subunit is important for IL-12 bioactivity. When Lieschke et al. constructed a scIL-12, the order of the subunits was found to affect the IL-12 biologic activity (45). When the p35 subunit came before the p40 subunit, there was greatly decreased IL-12 activity; in contrast, when the subunits were reversed, with p40 in front of p35, the scIL-12 had biologic activity comparable to rIL-12 (45, 46, 47). Similarly, in an OVA-IL-12 fusion protein in which the p40 subunit was fused to OVA, a 50-fold lower IL-12 activity was observed (48). Constraint of the p40 subunit in a fusion protein may disrupt the interaction between IL-12 and the IL-12R. The IL-12R complex consists of two chains, ß1 and ß2, with ß1 necessary for hIL-12 signaling and activity (49). It is thought that IL-12 interacts with the hIL-12R ß1 primarily through domains on the p40 subunit (50).
Given the need for an accessible p40 subunit, we chose to fuse the
scIL-12 to the amino terminus of the H chain. We were concerned that if
we fused the mIL-12.p40.linker.p35 to the carboxyl terminus of the
Ab H chain, we would constrain the p40 subunit and lose IL-12 activity.
In previous studies, it was found that both nerve growth factor (51)
and B7.1 (36) had to be joined to the amino terminus of the Ab to
maintain their activity in Ab fusion proteins; fusion at the carboxyl
terminus of the H chain resulted in impaired activity in both cases. We
find the IL-12 in our Ab-IL-12 fusion protein to be fully functional
with IL-12 bioactivity comparable to rIL-12.
Our studies contrast with the work of Gilles et al., who fused the p35 subunit to the carboxyl terminus of the H chain and expressed the p40 subunit from a separate vector (32). While this approach led to the production of functional fusion proteins, the IL-12 had only one-half of the expected bioactivity. In contrast to the single-chain approach, this approach requires the separate transfection of the two IL-12 subunits and does not guarantee that they are present in equimolar concentrations. Although the p40 subunit was not fused to the Ab in Ab-IL-12 fusion protein produced by Gilles et al., the fusion of the p35 subunit to the carboxyl terminus of the Ab without any type of flexible linker may make the p40 subunit somewhat less accessible for receptor binding; this could explain the 2-fold lower IL-12 activity they observed.
Both scIL-12 (75 kDa) and H chain (60 kDa) are large. However, by
providing a flexible linker between the two polypeptides, we were able
to maintain the activity of both. The presence of IL-12 at the amino
terminus of the VH region does not sterically
hinder the ability of the combining site of the Ab to interact with Ag
on the cell surface and remain bound (see Figs. 3
and 4). Similarly,
the IL-12 in the fusion protein appears to be unaffected in its ability
to bind the IL-12R and exhibit IL-12-mediated cellular activation
(
Figs. 5–7). The attachment of the IL-12 to the V region of the Ab
should position it near the surface of the tumor cell and may further
potentiate the antigenicity of the targeted tumor.
The ultimate goal of the construction of mscIL-12.her2.IgG3 is its use
as an antitumor agent. Using a CT26/Her2 tumor model previously
developed in our laboratory (33), our initial in vivo studies
demonstrate that this fusion protein has significant antitumor activity
in immunocompetent BALB/c mice (Fig. 8). We observed better antitumor
activity when treatment was started after the tumors were established
with a mean diameter of 8–9 mm than when treatment was started the day
after inoculation with tumor cells. This lends support to previous
studies by others (4, 52, 53) in which better antitumor activity of
IL-12 was observed when tumors were established. They proposed that
this may be because effector cells are first recruited to the tumor
site and are then activated by IL-12. Further work is being conducted
to determine whether the in vivo efficacy we have observed is due to
activated T or NK cells, whether a Th1 response has been stimulated,
and whether any other antitumor activities may have been stimulated by
treatment with mscIL-12.her2.IgG3.
In conclusion, we have demonstrated that it is possible to genetically engineer and express a scIL-12-Ab fusion protein that retains Her2/neu Ag specificity and IL-12 biologic activity comparable to rIL-12. Our results indicate that the bulky size of IL-12 does not affect Ag binding and that the Ab does not hinder cytokine receptor binding. Further, this fusion protein demonstrates antitumor activity in a tumor model using CT26/Her2 cells in syngeneic immunocompetent BALB/c mice. Thus, this Ab-IL-12 fusion protein may be an effective alternate to systemic administration of IL-12 for the treatment of metastatic breast cancer. Using the tumor-targeting ability of the Ab, it should be able to achieve effective local IL-12 concentration at the sites of tumors and metastases with lower doses of IL-12, thus decreasing the risk of toxicity associated with IL-12 treatment. An anti-Her2/neu mAb has had success in clinical trials for the treatment of Her2/neu-expressing metastatic breast cancer (20). Fusion of a cytokine-like IL-12 that has antitumor and antimetastatic properties to a Her2/neu-specific Ab may enhance its efficacy, particularly if it elicits a tumor-specific immune response.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Sherie L. Morrison, Department of Microbiology, Immunology, and Molecular Genetics, University of California, Los Angeles, 405 Hilgard Avenue, Los Angeles, CA 90095-1489. E-mail address:
3 Abbreviations used in this paper: msc, murine single-chain; m, murine; h, human; sc, single-chain; MTS, 3-(4, 5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)- 2-(4-sulfophenyl)-2H-tetrazolium, inner salt; PMS, phenazine methosulfate; PI, propidium iodide.
Received for publication November 24, 1998. Accepted for publication April 8, 1999.
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References |
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production. J. Immunol. 153:1697.[Abstract]
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RI, generates LAK activity and shows enhanced binding to the high affinity IL-2R. Immunotechnology 1:95.[Medline]
, M. N. Margolies, R. J. Ridge, R. E. Bruccoleri, E. Haber, R. Crea, et al 1988. Protein engineering of Ab binding sites: recovery of specific activity in an anti-digoxin single-chain Fv analogue produced in Escherichia coli. Proc. Natl. Acad. Sci. USA 85:5879. production by anti-tumor T cells. Int. Immunol. 7:1135.This article has been cited by other articles:
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  T.-H. Huang, K. R. Chintalacharuvu, and S. L. Morrison Targeting IFN-{alpha} to B Cell Lymphoma by a Tumor-Specific Antibody Elicits Potent Antitumor Activities J. Immunol., November 15, 2007; 179(10): 6881 - 6888. [Abstract] [Full Text] [PDF]
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 C. R. Ferrone, M.-A. Perales, S. M. Goldberg, C. J. Somberg, D. Hirschhorn-Cymerman, P. D. Gregor, M. J. Turk, T. Ramirez-Montagut, J. S. Gold, A. N. Houghton, et al. Adjuvanticity of plasmid DNA encoding cytokines fused to immunoglobulin fc domains. Clin. Cancer Res., September 15, 2006; 12(18): 5511 - 5519. [Abstract] [Full Text] [PDF]
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 C. F. Eisenbeis, G. B. Lesinski, M. Anghelina, R. Parihar, D. Valentino, J. Liu, P. Nadella, P. Sundaram, D. C. Young, M. Sznol, et al. Phase I Study of the Sequential Combination of Interleukin-12 and Interferon Alfa-2b in Advanced Cancer: Evidence for Modulation of Interferon Signaling Pathways by Interleukin-12 J. Clin. Oncol., December 1, 2005; 23(34): 8835 - 8844. [Abstract] [Full Text] [PDF]
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N. K. Dakappagari, J. Pyles, R. Parihar, W. E. Carson, D. C. Young, and P. T. P. Kaumaya A Chimeric Multi-Human Epidermal Growth Factor Receptor-2 B Cell Epitope Peptide Vaccine Mediates Superior Antitumor Responses J. Immunol., April 15, 2003; 170(8): 4242 - 4253. [Abstract] [Full Text] [PDF]
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E. M. Yoo, L. A. Wims, L. A. Chan, and S. L. Morrison Human IgG2 Can Form Covalent Dimers J. Immunol., March 15, 2003; 170(6): 3134 - 3138. [Abstract] [Full Text] [PDF]
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O. Christ, S. Matzku, C. Burger, and M. Zöller Interleukin 2-Antibody and Tumor Necrosis Factor-Antibody Fusion Proteins Induce Different Antitumor Immune Responses in Vivo Clin. Cancer Res., May 1, 2001; 7(5): 1385 - 1397. [Abstract] [Full Text]
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M. Imboden, K. R. Murphy, A. L. Rakhmilevich, Z. C. Neal, R. Xiang, R. A. Reisfeld, S. D. Gillies, and P. M. Sondel The Level of MHC Class I Expression on Murine Adenocarcinoma Can Change the Antitumor Effector Mechanism of Immunocytokine Therapy Cancer Res., February 1, 2001; 61(4): 1500 - 1507. [Abstract] [Full Text]
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J. S. Dela Cruz, K. R. Trinh, S. L. Morrison, and M. L. Penichet Recombinant Anti-Human HER2/neu IgG3-(GM-CSF) Fusion Protein Retains Antigen Specificity and Cytokine Function and Demonstrates Antitumor Activity J. Immunol., November 1, 2000; 165(9): 5112 - 5121. [Abstract] [Full Text] [PDF]
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 R. L. Shields, A. K. Namenuk, K. Hong, Y. G. Meng, J. Rae, J. Briggs, D. Xie, J. Lai, A. Stadlen, B. Li, et al. High Resolution Mapping of the Binding Site on Human IgG1 for Fcgamma RI, Fcgamma RII, Fcgamma RIII, and FcRn and Design of IgG1 Variants with Improved Binding to the Fcgamma R J. Biol. Chem., February 23, 2001; 276(9): 6591 - 6604. [Abstract] [Full Text] [PDF]
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