HER2/neu is a proto-oncogene that encodes a cell-sur-
face transmembrane receptor with intracellular tyro-
sine kinase activity (1). Upon ligand binding, the HER2
receptor dimerizes with other members of the EGF-R
family and initiates intracellular signals that result in
proliferation. The HER2/neu gene is amplified in
approximately 25% of human adenocarcinomas (2).
Overexpression of this receptor leads to ligand-inde-
pendent activation of the HER2 receptor kinase and
aberrant epithelial cell proliferation. Breast cancer
patients with this alteration frequently exhibit a worse
histological grade, decreased relapse-free and overall
survival, and altered sensitivity to standard chemother-
apeutic regimens (1, 3). A murine mAb directed against
the extracellular domain of the HER2 receptor
(mu4D5) was developed in the hope that tumors
exhibiting HER2-dependent growth might respond
with reduced proliferation (4). Indeed, this Ab does
inhibit the growth of HER2-positive tumor cells in
vitro and mediates regression of established tumors in
a variety of animal models (5). Therapy with a recom-
binant, humanized form of this Ab (trastuzumab, trade
name Herceptin) has shown efficacy in clinical trials,
both as a single agent and when administered with
chemotherapeutic drugs (6, 7). However, only 25–30%
of patients with HER2-overexpressing malignancies
will respond to these treatments. The ability to effec-
tively combine Herceptin with other anti-tumor agents
might be enhanced with a more complete understand-
ing of its mechanism of action.
The binding of Herceptin to the HER2 receptor may
lead to alterations in intracellular signaling and cell
cycle kinetics within the target cell (8). In addition,
Herceptin may also stimulate important immune
responses such as Ab-dependent cellular cytotoxicity
(ADCC) and complement activation that may lead to
direct tumor lysis (9). Indeed, it has been shown in a
murine tumor xenograft model that the anti-tumor
effects of Herceptin are dependent upon the presence
of host immune cells that express receptors for the Fc
portion of IgG (FcR) (10). Important ADCC-mediating
effector cells that express FcR include mono-
cytes/macrophages, resting and activated granulocytes,
and NK cells. Although most immune cells coexpress
both activating and inhibitory FcR’s, NK cells are
unique in that they constitutively express only the acti-
vating, low-affinity receptor FcγRIII (11). They also
contain abundant cytolytic granules, prominently
express cellular adhesion molecules, and constitutive-
ly display multiple cytokine receptors. Indeed, NK cell
cytotoxicity against HER2-expressing tumor cells is
The Journal of Clinical Investigation| October 2002| Volume 110| Number 7
IL-12 enhances the natural killer cell cytokine response
to Ab-coated tumor cells
Robin Parihar,1,2Julie Dierksheide,3Yan Hu,2and William E. Carson1,2,3
1Department of Molecular Virology, Immunology, and Medical Genetics,
2The Arthur G. James Comprehensive Cancer Center, and
3Department of Surgery, The Ohio State University College of Medicine, Columbus, Ohio, USA
The anti-tumor activity of recombinant mAb’s directed against tumor cell growth receptors has gen-
erally been considered to result from direct antiproliferative effects, the induction of apoptosis, or
possibly Ab-dependent cellular cytotoxicity mediated against tumor targets. However, it remains
unclear to what degree these mechanisms actually aid in the clearance of Ab-coated tumor cells in
vivo. We show here that NKcells secrete a distinct profile of potent immunostimulatory cytokines in
response to dual stimulation with Ab-coated tumor cells and IL-12. This response could not be dupli-
cated by costimulation with other ILs and was significantly enhanced in the presence of monocytes.
Cytokine production was dependent upon synergistic signals mediated by the activating receptor for
the Fc portion of IgG (FcγRIII) and the IL-12 receptor expressed on NK cells. Coadministration of
Ab-coated tumor cells and IL-12 to BALB/c mice resulted in enhanced circulating levels of NK
cell–derived cytokines with the capacity to augment anti-tumor immunity. These findings suggest
that, in addition to mediating cellular cytotoxicity and apoptosis, the anti-tumor activity of mAb’s
might also result from activation of a potent cytokine secretion program within immune effectors
capable of recognizing mAb-coated targets.
J. Clin. Invest. 110:983–992 (2002). doi:10.1172/JCI200215950.
Received for publication May 17, 2002, and accepted in revised form
August 30, 2002.
Address correspondence to: William E. Carson, N924 Doan
Hall, 410 West 10th Avenue, The Ohio State University College of
Medicine, Columbus, Ohio 43210, USA. Phone: (614) 293-6306;
Fax: (614) 688-4366; E-mail: email@example.com.
Conflict of interest: No conflict of interest has been declared.
Nonstandard abbreviations used: Ab-dependent cellular
cytotoxicity (ADCC); macrophage inflammatory protein-1α
(MIP-1α); antigen presenting cell (APC); IL-12 receptor (IL-12R);
recombinant human (rhu); recombinant murine (rmu);
phycoerythrin (PE); protein tyrosine kinase (PTK).
markedly enhanced following treatment with IL-2 or
IL-12 (12, 13). NK cells are also sources of potent
immunostimulatory cytokines such as TNF-α,
GM-CSF, macrophage inflammatory protein-1α(MIP-
1α), and IFN-γ(11). Thus, while the anti-tumor activi-
ty of Herceptin has largely been attributed to direct
antiproliferative and proapoptotic effects, we theorized
that the clearance of Ab-coated tumor cells might be
enhanced by the coadministration of immunologic
adjuvants with the capacity to stimulate NK cell cyto-
toxicity and cytokine production.
IL-12 is an antigen presenting cell–derived (APC-
derived) cytokine that stimulates T cells and NK cells to
secrete IFN-γ and augments the proliferation and
cytolytic activity of these cells (14). In addition to its crit-
ical role in the regulation of early inflammatory respons-
es and promotion of the Th1-type repertoire, preclinical
studies have suggested that IL-12 is an effective anti-can-
cer agent against various experimental malignancies.
Work in numerous murine models has demonstrated
that the anti-tumor effects of IL-12 are primarily medi-
ated via the induction of IFN-γ secretion by T and NK
cells (15, 16). Of note, IL-12–induced IFN-γ production
has been shown to prevent mammary carcinogenesis in
HER2/neutransgenic mice when IL-12 was administered
alone or in combination with tumor cell– or dendritic
cell–based vaccines (17, 18). Neutralization of IFN-γ in
this and other models strongly inhibits the anti-tumor
activity of IL-12. Similarly, depletion of NK cells within
hosts given exogenous IL-12 has been shown to attenu-
ate the anti-tumor effect, suggesting a vital role for NK
cells in the anti-tumor activity of IL-12 (19).
In the current report, we have examined the ability of
IL-12 to enhance NK cell–mediated cytotoxicity and
promote immunostimulatory cytokine secretion. We
demonstrate that costimulation of pure NK cells or
whole PBMCs with Herceptin-coated human breast
cancer cells and IL-12 results in a unique cytokine secre-
tion profile highlighted by abundant production of
IFN-γ. Furthermore, we provide evidence that cytokine
production in response to Herceptin-coated tumor cells
and IL-12 is mediated by synergistic signals provided by
FcγRIII and the IL-12 receptor (IL-12R), and that this
synergistic effect could not be duplicated by IL-2, IL-10,
IL-15, or IL-18. These findings provide a strong ration-
ale for the concurrent administration of Herceptin and
IL-12 for the treatment of HER2-overexpressing malig-
nancies and lend insight into the potential role of NK
cells in the elimination of Ab-coated tumor targets.
Cytokines and Ab’s. Recombinant human IL-12 (rhuIL-
12) and murine IL-12 (rmuIL-12) were kindly provided
by Genetics Institute Inc. (Cambridge, Massachusetts,
USA). rhuIL-2 with a specific activity of 1.53 × 107
U/mg was obtained from Hoffman-LaRoche Inc.
(Nutley, New Jersey, USA). rhuIL-15, rhuIL-18, and
rhuIL-10 were purchased from PeproTech Inc. (Rocky
Hill, New Jersey, USA). Cytokines were resuspended in
1× PBS plus 0.1% BSA. The humanized anti-HER2
mAb Herceptin was kindly provided by Genentech Inc.
(San Francisco, California, USA) (20). Purified F(ab′)2
fragments of Herceptin were generated by pepsin diges-
tion as previously described (21). Polyclonal huIgG was
purchased from Sigma-Aldrich (St. Louis, Missouri,
USA). For preliminary costimulation experiments, 96-
well flat-bottom plates were coated with huIgG by
incubation with 100 µg/ml huIgG in 100 µl of PBS
overnight at 4°C. Wells were then washed twice with
200 µl fresh PBS and once with warm 10% HAB medi-
um consisting of heat-inactivated pooled human AB
serum (C-six Diagnostics Inc., Germantown, Wiscon-
sin, USA), 100 U/ml penicillin, 100 µg/ml streptomy-
ocin, and 0.25 µg/ml amphotericin, prior to use.
Cell lines. The SKBR3 (HER2-overexpressing) and
MDA-468 (HER2-negative) human breast adenocarci-
noma lines were obtained from American Type Culture
Collection (Manassas, Virginia, USA). The MCF-
7HER2/neu(HER2-overexpressing) human breast adeno-
carcinoma was obtained from Jose Baselga (Hospital
Universitari Vall D’Hebron, Barcelona, Spain). The
MCF-7HER2/neucell line was created by transfection of
parental MCF-7 cells with an expression cassette for
human HER2/neu and culture in standard medium
containing neomycin (22). A murine colon carcinoma
line overexpressing human HER2/neu, CT-26HER2/neu,
and the parental line, CT-26, were kind gifts from P.T.P.
Kaumaya (Ohio State University, Columbus, Ohio,
USA) and were used for in vivo costimulation studies
(23). Cancer cell lines were propagated in RPMI-1640
medium supplemented with 10% heat-inactivated FBS,
300 µg/ml L-glutamine, 100 U/ml penicillin, 100 µg/ml
streptomycin, 0.25 µg/ml amphotericin B, and 0.06
mg/ml anti-PPLO agent (all from Life Technologies
Inc., Rockville, Maryland, USA).
Mice. Female BALB/c mice between the ages of 5 and
7 weeks were purchased from The Jackson Laboratory
(Bar Harbor, Maine, USA). STAT4–/–breeder mice on a
B6 × 129 strain background were a kind gift from
James Ihle (St. Jude Children’s Research Hospital,
Memphis, Tennessee, USA), and females were used in
experiments at 5–7 weeks of age (24). Age-matched
wild-type B6×129 mice (The Jackson Laboratory) were
used as controls. Mice were maintained in the animal
facility at the Ohio State University Comprehensive
Cancer Center (CCC) with free access to food and
water. All protocols were approved by the Ohio State
University CCC Animal Care and Use Committee, and
mice were treated in accordance with the institutional
guidelines for animal care.
Isolation of human NK cells. PBMCs were isolated from
fresh leukopacks (American Red Cross, Columbus,
Ohio, USA) using Ficoll-Hypaque (Sigma-Aldrich) den-
sity gradient centrifugation. PBMCs were washed in
RPMI-1640 (Life Technologies Inc.) and adhered
overnight to plastic plates to eliminate the monocyte
population. Nonadherent peripheral blood lympho-
cytes were further depleted of T cells, B cells, and mono-
The Journal of Clinical Investigation| October 2002| Volume 110| Number 7
pressing metastatic breast cancer refractory to chemotherapy treatment.
J. Clin. Oncol. 16:2659–2671.
8.Baselga, J., and Albanell, J. 2001. Mechanisms of action of anti-HER2
monoclonal antibodies. Ann. Oncol. 12:S35–S41.
9.Sliwkowski, M.X., et al. 1999. Nonclinical studies addressing the mech-
anism of action of trastuzumab (Herceptin). Semin. Oncol. 26:60–70.
10.Clynes, R.A., Towers, T.L., Presta, L.G., and Ravetch, J.V. 2000. Inhibito-
ry Fc receptors modulate in vivo cytotoxicity against tumor targets. Nat.
11.Robertson, M.J., and Ritz, J. 1990. Biology and relevance of human nat-
ural killer cells. Blood. 76:2421–2438.
12.Carson, W.E., et al. 2001. Interleukin-2 enhances the natural killer cell
response to Herceptin-coated Her2/neu-positive breast cancer cells. Eur.
J. Immunol. 31:3016–3025.
13.Lieberman, M.D., Sigal, R.K., Williams, N.N., and Daly, J.M. 1991. Nat-
ural killer cell stimulatory factor (NKSF) augments natural killer cell
antibody-dependent tumoricidal response against colon carcinoma cell
lines. J. Surg. Res. 50:410–415.
14.Gately, M.K., et al. 1998. The interleukin-12/interleukin-12 receptor sys-
tem: role in normal and pathologic immune responses. Ann. Rev.
15.Tannenbaum, C.S., et al. 1996. Cytokine and chemokine expression in
tumors of mice receiving systemic therapy with IL-12. J. Immunol.
16.Haicheur, N., et al. 2000. Cytokines and soluble cytokine receptor induc-
tion after IL-12 administration in cancer patients. Clin. Exp. Immunol.
17.Nanni, P., et al. 2001. Combined allogeneic tumor cell vaccination and
systemic IL-12 prevents mammary carcinogenesis in HER-2/neu trans-
genic mice. J. Exp. Med. 194:1195–1205.
18.Chen, Y., et al. 2001. Induction of ErbB2/neu-specific protective and
therapeutic anti-tumor immunity using genetically modified dendritic
cells: enhanced efficacy by co-transduction of gene encoding IL-12. Gene
19.Yao, L., et al. 1999. Contribution of natural killer cells to inhibition of
angiogenesis by interleukin-12. Blood. 93:1612–1621.
20.Carter, P., et al. 1992. Humanization of an anti-p185HER2antibody for
human cancer therapy. Proc. Natl. Acad. Sci. USA. 89:4285–4289.
21.Jones, R.G., and Landon, J. 2002. Enhanced pepsin digestion: a novel
process for purifying antibody F(ab′)2 fragments in high yield from
serum. J. Immunol. Methods. 263:57–74.
22.Liu, Y., el-Ashry, D., Chen, D., Ding, I.Y., and Kern, F.G. 1995. MCF-7
breast cancer cells overexpressing transfected c-erbB-2 have an in vitro
growth advantage in estrogen-depleted conditions and reduced estro-
gen-dependence and tamoxifen-sensitivity in vivo. Breast Cancer Res. Treat.
23.Penichet, M.L., et al. 1999. In vivo properties of three human HER2/neu-
expressing murine cell lines in immunocompetent mice. Lab. Anim. Sci.
24.Thierfelder, W.E., et al. 1996. Requirement for Stat4 in interleukin-12-
mediated responses of natural killer and T cells. Nature. 382:171–174.
25.Carson, W.E., et al. 1994. Interleukin-15 is a novel cytokine which acti-
vates human natural killer cells via components of the interleukin-2
receptor. J.Exp. Med. 180:1395–1403.
26.Andersen, B.L., et al. 1998. Stress and immune responses after surgical
treatment for regional breast cancer. J. Natl. Cancer Inst. 90:30–36.
27.Scott, S.D. 1998. Rituximab: a new therapeutic monoclonal antibody for
non-Hodgkin’s lymphoma. Cancer Pract. 6:195–197.
28.Oliver, J.M., Burg, D.L., Wilson, B.S., McLaughlin, J.L., and Geahlen, R.L.
1994. Inhibition of mast cell Fc epsilon R1-mediated signaling and effec-
tor function by the Syk-selective inhibitor, piceatannol. J. Biol. Chem.
29.Cooper, M.A., Fehniger, T.A., and Caligiuri, M.A. 2001. The biology of
human natural killer-cell subsets. Trends Immunol. 22:633–640.
30.Cassatella, M.A., et al. 1989. Fc gamma R(CD16) interaction with ligand
induces Ca2+ mobilization and phosphoinositide turnover in human
natural killer cells. Role of Ca2+ in Fc gamma(CD16)-induced tran-
scription and expression of lymphokine genes. J. Exp. Med. 169:549–567.
31.Sahin, U., Kraft-Bauer, S., Ohnesorge, S., Pfreundschuh, M., and Renner,
C. 1996. Interleukin-12 increases bispecific-antibody-mediated natural
killer cell cytotoxicity against human tumors. Cancer Immunol.
32.Aste-Amezaga, M., D’Andrea, A., Kubin, M., and Trinchieri, G. 1994.
Cooperation of natural killer cell stimulatory factor/interleukin-12 with
other stimuli in the induction of cytokines and cytotoxic cell-associated
molecules in human T and NK cells. Cell. Immunol. 156:480–492.
33.Bonnema, J.D., et al. 1994. Cytokine-enhanced NK cell-mediated cyto-
toxicity: positive modulatory effects of IL-2 and IL-12 on stimulus-
dependent granule exocytosis. J. Immunol. 152:2098–2104.
34.Ortaldo, J.R., Mason, A.T., and O’Shea, J.J. 1995. Receptor-induced death
in human natural killer cells: involvement of CD16. J. Exp. Med.
35.Gobin, S.J., and van den Elsen, P.J. 2000. Transcriptional regulation of
the MHC class Ib genes HLA-E, HLA-F and HLA-G. Hum. Immunol.
36.Yoon, S.J., et al. 1998. Synergistic anti-tumor effects with co-expression
of GM-CSF and IFN-gamma in tumors. Int. J. Cancer. 77:907–912.
37.Prevost-Blondel, A., Roth, E., Rosenthal, F.M., and Pricher, H. 2000. Cru-
cial role of TNF-alpha in CD8 T cell-mediated elimination of 3LL-A9
Lew carcinoma cells in vivo. J.Immunol. 164:3645–3651.
38.Walker, W., Aste-Amezaga, M., Kastelein, R.A., Trinchieri, G., and
Hunter, C.A. 1999. IL-18 and CD28 use distinct molecular mechanisms
to enhance NK cell production of IL-12-induced IFN-gamma.
J. Immunol. 162:5894–5901.
39.Cheung, J.C., Koh, C.Y., Gordon, B.E., Wilder, J.A., and Yuan, D. 1999.
The mechanism of activation of NK-cell IFN-gamma production by lig-
ation of CD28. Mol. Immunol. 36:361–372.
40.Ting, A.T., et al. 1995. Interaction between lck and syk family tyrosine
kinases in Fc gamma receptor-initiated activation of natural killer cells.
J. Biol. Chem. 270:16415–16421.
41.Pignata, C., et al. 1995. Phosphorylation of src family lck tyrosine kinase
following interleukin-12 activation of human natural killer cells. Cell.
42.Manciulea, M., et al. 1996. Divergent phosphotyrosine signaling via
FcγRIIIA on human NK cells. Cell. Immunol. 167:63–71.
43.Yoshida, A., et al. 2001. IL-18-induced expression of intercellular adhe-
sion molecule-1 in human monocytes: involvement in IL-12 and
IFN-gamma production in PBMC. Cell. Immunol. 210:106–115.
44.Tominaga, K., et al. 2000. IL-12 synergizes with IL-18 or IL-1beta for
IFN-gamma production from human T cells. Int. Immunol. 12:151–160.
45.Skok, J., Poudrier, J., and Gray, D. 1999. Dendritic cell-derived IL-12 pro-
motes B cell induction of Th2 differentiation: a feedback regulation of
Th1 development. J. Immunol. 163:4284–4291.
46.Amakata, Y., et al. 2001. Mechanism of NK cell activation induced by
coculture with dendritic cells derived from peripheral blood monocytes.
Clin. Exp. Immunol. 124:214–222.
47.Chace, J.H., Hooker, N.A., Mildenstein, K.L., Krieg, A.M., and Cowdery,
J.S. 1997. Bacterial DNA-induced NK cell IFN-gamma production is
dependent on macrophage secretion of IL-12. Clin. Immunol.
48.Rodriguez-Calvillo, M., et al. 2002. Upregulation of natural killer cells
functions underlies the efficacy of intratumorally injected dendritic cells
engineered to produce interleukin-12. Exp. Hematol. 30:195–204.
49.van Ojik, H.H., Repp, R., Groenewegen, G., Valerius, T., and van de
Winkel, J.G. 1997. Clinical evaluation of the bispecific antibody MDX-
H210 (anti-Fc gamma RI x anti-HER-2/neu) in combination with gran-
ulocyte-colony-stimulating factor (filgrastim) for treatment of advanced
breast cancer. Cancer Immunol. Immunother. 45:207–209.
50.Fleming, G.F., et al. 1999. A phase I CALGB trial of recombinant human
anti-HER2 monoclonal antibody plus low-dose interleukin2 in solid
tumors. Proc. Am. Soc. Clin. Oncol. 18:184a. (Abstr. 710)
51.Shalaby, M.R., et al. 1992. Development of humanized bispecific anti-
bodies reactive with cytotoxic lymphocytes and tumor cells overexpress-
ing the HER2 protooncogene. J. Exp. Med. 175:217–225.
52.Weiner, L.M., et al. 1995. Phase I trial of 2B1, a bispecific monoclonal
antibody targeting c-erbB-2 and Fc gamma RIII. Cancer Res.
53.Valone, F.H., et al. 1995. Phase Ia/Ib trial of bispecific antibody MDX-
210 in patients with advanced breast or ovarian cancer that overexpresses
the proto-oncogene HER-2/neu. J. Clin. Oncol. 13:2281–2292.
54.Gerstmayer, B., Hoffmann, M., Altenschmidt, U., and Wels, W. 1997. Co-
stimulation of T-cell proliferation by a chimeric B7-antibody fusion pro-
tein. Cancer Immunol. Immunother. 45:156–158.
55.Penichet, M.L., Dela Cruz, J.S., Shin, S.U., and Morrison, S.L. 2001. A
recombinant IgG3-(IL-2) fusion protein for the treatment of human
HER2/neu expressing tumors. Hum. Antibodies. 10:43–49.
56.Dela Cruz, J.S., Trinh, K.R., Morrison, S.L., and Penichet, M.L. 2000.
Recombinant anti-human HER2/neu IgG3-(GM-CSF) fusion protein
retains antigen specificity and cytokine function and demonstrates anti-
tumor activity. J. Immunol. 165:5112–5121.
57.Peng, L.S., Penichet, M.L., and Morrison, S.L. 1999. A single-chain IL-12
IgG3 antibody fusion protein retains antibody specificity and IL-12
bioactivity and demonstrates antitumor activity. J. Immunol.
58.Cavallo, F., et al. 1999. Immune events associated with the cure of estab-
lished tumors and spontaneous metastases by local and systemic inter-
leukin-12. Cancer Res. 59:414–421.
59.Parihar, R. et al. 2001. A phase I trial of Herceptin and interleukin-12 in
patients with HER2-overexpressing malignancies. Proc. Am. Soc. Clin.
Oncol. 20:258a. (Abstr. 1031)
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