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RI, CD89) and Tumor Antigens Efficiently Promote Cell-Mediated Cytotoxicity of Tumor Targets in Whole Blood
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Medarex, Inc., Annandale, NJ 08801; and
Department of Microbiology, Dartmouth Medical School, Lebanon, NH 03756
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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RI, CD89) is primarily expressed on cytotoxic
immune effector cells. By chemically cross-linking F(ab') fragments of
the FcR for IgA (FcRI)-specific mAb (A77) with tumor Ag-specific mAb
(anti-HER2/neu and anti-epidermal growth factor
receptor), we have developed bispecific molecules (BSM) that
simultaneously bind to respective tumor Ags and FcRI-expressing
effector cells in whole blood. These BSM mediated up to 55% of
specific lysis of appropriate tumor Ag-expressing target cells (from a
variety of tumors) with purified polymorphonuclear leukocytes,
monocytes, or whole blood effector cells without preactivation with
exogenous cytokines. To our knowledge, this is the first demonstration
of Ab-dependent cell-mediated cytotoxic activity via FcRI in whole
blood. Also, monocyte-derived macrophages mediated phagocytosis of
HER2/neu-expressing tumor cells (>95% tumor cell loss).
These BSM-mediated cytotoxic activities were completely inhibited by
F(ab')2 of A77, demonstrating the specific role of FcRI
as a trigger molecule. Furthermore, the binding of these BSM to
monocytes or polymorphonuclear leukocytes in whole blood did not induce
modulation of FcRI in the absence of the target Ag. Therefore,
immune effector cells may be "armed" with FcRI-directed BSM in
whole blood. These FcRI-directed BSM may offer new treatment options
for various malignancies and other disease conditions. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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RI2 or CD89, has
been identified and characterized (2, 3). FcRI is constitutively
expressed primarily on cytotoxic immune effector cells including
polymorphonuclear leukocytes (PMN), monocytes, macrophages,
neutrophils, and eosinophils (4). FcRI expression on a subpopulation
of lymphocytes (4) and glomerular mesangial cells has been reported
(5). Its expression on monocytes and PMN can be enhanced by TNF- (6, 7), IL-1, granulocyte macrophage-CSF, LPS, or phorbol esters (8, 9),
whereas IFN-
and TGF-ß1 decrease FcRI expression (10). The
-chain of human FcRI is a heavily glycosylated, type 1
transmembrane molecule belonging to the Ig supergene family, which also
includes receptors for IgG and IgE. One gene located on chromosome 19
encodes several alternatively spliced isoforms of the FcRI -chain
(55–110 kDa) (4). It has been shown previously that myelocytic FcRI
are associated with the FcR -chain that is thought to play a role in
FcRI signal transduction (11, 12, 13). FcRI binds both Ag-complexed
and monomeric IgA1 and IgA2 (14), suggesting that the receptor may be
saturated in vivo with monomeric IgA in the same manner that the FcR
for IgG (FcRI) and the FcR for IgE are saturated with IgG and IgE,
respectively. Cross-linking FcRI on myeloid effector cells by
polymeric IgA, IgA immune complexes, or mAbs specific for epitopes
within or outside the ligand-binding domain stimulates degranulation,
superoxide release, secretion of inflammatory cytokines, endocytosis,
and phagocytosis (15, 16, 17, 18). It is presumed that these physiologic
responses triggered via FcRI may be important in the first line of
humoral defense on mucosal surfaces (4). Thus, FcRI appears to be a
clinically relevant trigger receptor on cytotoxic immune effector
cells, and its activity can be exploited to develop novel
immunotherapies. However, to date the cytotoxic potential of FcRI
has not been carefully explored, since almost all mAb-based therapies
are being developed with mAbs of IgG class that do not bind to
FcRI.
To our knowledge, tumor-specific mAbs of human IgA class are not
available. Also, it is likely that serum IgA (up to 4.0 mg/ml)
interferes with the activity of IgA mAbs under physiologic conditions.
Therefore, we used another approach by employing bispecific molecules
(BSM) to study FcRI-dependent cell-mediated cytotoxicity of tumor
targets. BSM, which simultaneously bind to target cells (tumor cells,
pathogens) and a trigger receptor (e.g., CD3, CD2, FcRI) on immune
effector cells, have been previously described (19, 20). BSM can be
generated from heterohybridomas or by chemically or genetically linking
the F(ab') fragments of two Abs with different specificities or an
F(ab') fragment and a ligand (21, 22). BSM that is produced using a
trigger receptor-specific Ab, which binds outside the natural
ligand-binding domain of the trigger receptor, can circumvent
interference by serum Abs and recruit immune effector cells in the
presence of a saturating concentration of the natural ligand (23). This
strategy has been used to produce FcRI-specific BSM, which promote
Ab-dependent cell-mediated cytotoxity (ADCC) of tumor cells in the
presence of monomeric or aggregated IgG (19) and have shown promising
results in clinical settings (24). Four FcRI-specific mAbs that bind
outside the IgA ligand-binding domain have been described previously
(25). Using one of these mAbs (A77), we developed BSM by chemically
linking the F(ab') of A77 to the F(ab') of anti-tumor mAbs specific
for HER2/neu or epidermal growth factor receptor (EGF-R).
Both HER2/neu and EGF-R are well-characterized
tumor-associated Ags that are overexpressed on the surface of a variety
of tumors and therefore provide a suitable target for specific tumor
recognition. Several immunotherapies targeted to these two
tumor-associated Ags are being developed (24). The biochemical,
immunologic, and functional characterization of these FcRI-directed
BSM and the potential applications of FcRI-directed immunotherapies
are described in this report.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The anti-FcRI mAb, A77, the anti-HER2/neu
mAb (520C9), and the anti-EGF-R mAb (H425) were purified from
respective cell culture supernatants by protein A chromatography. The
A77- and 520C9-producing murine hybridomas have been described
previously (25, 26). The genetic construct for the humanized
anti-EGF-R mAb (H425) and its transient expression in COS cells has
also been described previously (27). This genetic construct was
transfected into a nonsecretory myeloma cell line (NS 0) to stably
produce H425 mAb. This H425 mAb-producing cell line was generously
provided by Dr. W. Strittmatter of Merck KGaA (Darmstadt, Germany). All
of the mAb-producing cell lines were cultivated in Iscove’s modified
Dulbecco’s medium (Life Technologies, Grand Island, NY) supplemented
with 10% FBS. SKBR-3 and SKOV-3 cell lines that overexpress
HER2/neu proto-oncogene, as well as A431 and MDA-MB-468 cell
lines that overexpress EGF-R, were obtained from the American Type
Culture Collection (Rockville, MD). Another EGF-R-overexpressing cell
line, LICR-LON-HN5 (HN5), was kindly provided by Dr. C. J. Dean
from the Institute of Cancer Research (Sutton, U.K). All of the tumor
cell lines were cultivated in Iscove’s modified Dulbecco’s medium
supplemented with 10% FBS.
BSM coupling procedure
BSM were constructed using the method of Glennie et al. (28). mAbs A77, 520C9, or H425 were separately digested with pepsin to F(ab')2 and subsequently reduced to Fab' by the addition of 10 mM mercaptoethylamine for 30 min at 30°C. The Fab' fragments were applied to a Sephadex G-25 column (Pharmacia, Piscataway, NJ) equilibrated in 50 mM sodium acetate, 0.5 mM EDTA, pH 5.3 (4°C). To produce A77 x 520C9 BSM, ortho-phenylenedimaleimide (o-PDM) (12 mM) that had dissolved in dimethylformamide and chilled in a methanol/ice bath was added (one-half volume) to the 520C9 Fab' and incubated for 30 min on ice. The Fab'-maleimide was then separated from free o-PDM on a Sephadex G-25 column equilibrated in 50 mM sodium acetate and 0.5 mM EDTA, pH 5.3 (4°C). The 520C9 Fab'-maleimide was added to A77 Fab' at a 1:1 M ratio. The reactants were concentrated using nitrogen to the starting volume using a Diaflo membrane (Amicon, Beverly, MA) in an Amicon chamber (all at 4°C). After 18 h, the pH was adjusted to 8.0 with 1 M Tris-HCl (pH 8.0). The mixture was then reduced with 10 mM mercaptoethylamine (30 min, 30°C) and alkylated with 25 mM iodoacetamide. The bispecific F(ab')2/F(ab')3 was separated from unreacted Fab and other products by a Superdex 200 column (Pharmacia, Piscataway, NJ) equilibrated in PBS. The A77 x H425 BSM was produced using the same method, except that A77 F(ab')/o-PDM derivative was initially prepared and was then conjugated with the H425 F(ab') as described above. All of the BSM preparations were tested for endotoxin contamination by chromogenic Limulus amebocyte lysate assay (BioWhittaker, Walkersville, MD) and all were found to be free of contamination (assay sensitivity limit 0.1 endotoxin unit/ml).
Binding by flow cytometry
BSM binding to FcRI, HER2/neu, or EGF-R was
assessed by flow cytometry. Various concentrations of BSM were diluted
in PBS (pH 7.4) containing 2 mg/ml BSA and 0.05% NaN3
(PBA) and were then incubated with either SKBR-3 or A431 cells for
1 h on ice. The cells were washed with PBA and incubated with
FITC-labeled goat anti-mouse or anti-human Ab for 1 h on
ice. Next, the cells were washed and fixed with 1% paraformaldehyde.
Cell-associated fluorescence was analyzed using a Becton Dickinson
FACScan (San Jose, CA). To examine BSM binding to FcRI and to
evaluate modulation of FcRI after BSM or A77 binding, various
concentrations of the respective Abs were added directly to the whole
blood. For binding assays, the blood was incubated at 4°C for 1
h with BSM. For modulation assays, the blood was incubated overnight at
37°C, 5% CO2. Erythrocytes were lysed by a lysis buffer
(1.7 M NH4Cl, 0.1 M KHCO3 and 1 mM EDTA)
(Becton Dickinson) and the remaining leukocytes were stained with
phycoerythrin12 (PE)-labeled goat anti-murine Ab for
binding assay or goat anti-human IgA-PE Ab for modulation assay.
The cells were washed, fixed with 1% paraformaldehyde, and
the lymphocyte, monocyte, or PMN-associated fluorescence was analyzed
using the FACScan. Lymphocyte, monocyte, or granulocyte populations
were gated using forward light scatter and side light scatter
parameters.
Ab-dependent cell-mediated cytotoxicity (ADCC)
HER2/neu-overexpressing SKBR-3 cells or EGF-R-overexpressing A431 cells were used as targets for lysis by heparinized whole blood, PMN purified from whole blood, or monocytes purified from leukopacs (Advanced Biotechnologies Inc., Columbia, MD) as previously described (29). These freshly purified PMN and monocytes were used as effector cells without any preactivation or cultivation. Targets were labeled with 100 µCi of 51Cr for 1 h before combining with effector cells and BSM in a U-bottom microtiter plate. After incubation for 16 to 18 h at 37°C, supernatants were collected and analyzed for radioactivity. BSM-dependent lysis of other EGF-R- or HER2/neu-overexpressing cell lines was examined similarly. Cytotoxicity was calculated by the formula: % lysis = (experimental cpm - target leak cpm/detergent lysis cpm - target leak cpm) x 100%. BSM-dependent lysis = % lysis with BSM - % lysis without BSM. Assays were performed in triplicate.
BSM-mediated phagocytosis
BSM-mediated phagocytosis of SKBR-3 cells by monocyte-derived macrophages (MDM) was examined by a modification of the method described by D. Munn and N.-K. Cheung (30). Briefly, monocytes that had been purified from normal adult source leukopacs (Advanced Biotechnologies Inc.) were differentiated in 24-well plates in macrophage serum-free medium (Life Technologies) supplemented with 10% FBS and 10 µg/ml of macrophage-CSF for 7 to 10 days. SKBR-3 cells were labeled with the lipophilic red fluorescent dye PKH 26 (Sigma, St. Louis, MO). The labeled SKBR-3 cells were added to the wells containing MDM in the absence or presence of BSM and incubated at 37°C for 24 h. MDM and nonphagocytized SKBR-3 cells were recovered with trypsin and stained with an FITC-labeled anti-CD14 mAb (AML-2-23) for 1 h on ice. Cells were washed and analyzed by two-color fluorescence using the FACScan. The percentage of phagocytosis was calculated as the number of dual-positive target cells (ingested by MDM) divided by the total number of target cells.
Confocal imaging
To confirm that the dual-positive events in the flow cytometric
phagocytic assay represented true phagocytosis of SKBR-3 by MDM,
dual-positive cells were sorted and examined by confocal microscopy.
After fixation and flow cytometric analysis, the dual-positive cells
were sorted with a FACStarPlus flow cytometer (Becton
Dickinson), centrifuged, and then placed on glass slides for imaging
with a Bio-Rad MRC1024 laser-scanning confocal microscope (Bio-Rad,
Hercules, CA). Cells were scanned for fluorescence using the 488-nm
line from a 15-mW KR/AR laser and two photodetectors (Bio-Rad)
(522/32-nm dichroic for FITC fluorescence and 585-nm longpass for
PKH-26 fluorescence). A 63x Plan-Apo 1.4 NA objective (Carl Zeiss,
Inc., Thornwood, NY) in conjunction with an iris setting of 2.5 allowed
for detection of optical sections of the fluorescence image that were
1.5 µm thick. Representative images were selected from the slices
through the center of each MDM after sectioning the entire
cell.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To determine the potential use of BSM for recruiting
FcRI-mediated cytotoxic activity, two BSM were constructed by
chemical conjugation of FcRI-specific mAb (A77) with two tumor
Ag-specific Abs that have been well characterized; one anti-tumor
Ab (520C9) specific for HER2/neu and the second specific for
EGF-R (H425). Analysis of A77 x 520C9 BSM by HPLC showed that
this BSM comprised two main species: 75 to 85% of F(ab')2
heterodimer (100 kDa, 10.43 min) and 15 to 25% of F(ab')3
heterotrimer (150 kDa, 9.86 min) (Fig. 1
). Based on the method of preparation,
the F(ab')3 species is believed to comprise two 520C9
F(ab') and one A77 F(ab'). The other BSM (A77 x H425) had similar
composition except that here the F(ab')3 species is
believed to comprise two A77 F(ab') and one H425 F(ab'). The m.w.
distribution was confirmed by SDS-PAGE analysis (data not shown) and is
consistent with that of an FcR-specific BSM prepared by the same
o-PDM linkage procedure (31). A control experiment in which
an F(ab')o-PDM derivative was incubated without a
nonderivatized second F(ab') confirmed that the F(ab')/o-PDM
derivative does not cross-link with itself, since all of the hinge
sulfhydryl groups are occupied by o-PDM and no
free-sulfhydryl group is available for linkage (data not shown). Thus,
the F(ab')2 and F(ab')3 species in these BSM
preparations are heterocomplexes of F(ab') fragments with two different
specificities. This dual specificity was examined by the determination
of binding activity using flow cytometry.
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shows that in whole blood,
A77 x 520C9 BSM binds to FcRI-expressing PMN and monocytes,
but not to FcR-negative lymphocytes. This binding activity was not
inhibited by physiologic levels of serum IgA, which are sufficient to
saturate all of the FcRI expressed in vivo. The binding specificity
of the tumor Ag-specific arm of A77 x 520C9 BSM to
HER2/neu-positive SKBR-3 cells is shown in Figure 3A. As expected, only the BSM
and 520C9 F(ab')2, and not A77 F(ab')2 control,
bound to these tumor cells. Similarly, Figure 3B shows that
the A77 x H425 and H425 F(ab')2, but not the A77
F(ab')2, bound to A431 cells.
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BSM-mediated destruction of tumor cells by FcRI-expressing
cytotoxic effector cells was examined using freshly purified effector
cells (monocytes and PMN) as well as whole blood as a source of
effector cells. Figure 4A
shows that A77 x 520C9 BSM mediated up to 37% of BSM-dependent
lysis of HER2/neu-positive SKBR-3 cells by purified PMN.
This cytotoxic activity was dose dependent and saturated at 1.0 µg/ml
of the BSM. Figure 4B shows that A77 x 520C9 also
mediated up to 40% of BSM-dependent lysis of SKBR-3 cells when
purified monocytes were used as effector cells. Finally, A77 x
520C9 BSM mediated up to 40% of BSM-dependent lysis of the same target
cells when whole blood was used as a source of effector cells (Fig. 4C). In all three experiments, A77
F(ab')2 inhibited the ADCC activity of this BSM, but the
anti-CD64 M22 F(ab')2 did not. The background lysis
(without BSM) in all the experiments was 10%.
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A), up to 55% of
BSM-dependent lysis with purified monocytes as effector cells (Fig. 5B), and up to 43% of BSM-dependent lysis with whole
blood as a source of effector cells (Fig. 5C). Again,
in all three experiments only A77 F(ab')2, and not M22
F(ab')2, inhibited the cytotoxic activity. The background
lysis (without BSM) was 10%. Experiments in which lower E:T ratios
were used showed that significant ADCC was achieved at an E:T ratio as
low as 12:1 (data not shown).
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MDM are known to mediate phagocytosis of tumor cells (32).
However, phagocytosis of tumor cells mediated by FcRI has not been
described. To determine whether A77 x 520C9 BSM could induce
phagocytosis, MDM were incubated with dye-labeled SKBR-3 cells in the
presence of varying concentrations of BSM. The level of phagocytosis
was determined by two-color flow cytometric analysis. Figure 6A shows that the MDM
(panel 1, FL1+, FL2-) and
SKBR-3 cells (panel 2, FL1-,
FL2+) are distinguished from each other in a mixture of
these two cell types by their unique fluorescence patterns
(panel 3). When the A77 x 520C9 BSM was
added to the mixture of these target and effector cells, the BSM
mediated nearly a complete loss of tumor cells (panel 5,
lower right quadrant). This was confirmed by an almost
total lack of tumor cells that could be recovered from the
BSM-containing wells as determined by tumor-specific ELISA (data not
shown). MDM alone mediated phagocytosis (53%) of SKBR-3 cells
without the BSM (panel 3). However, the
addition of 0.1 µg/ml A77 x 520C9 was sufficient to enhance the
phagocytosis to >95% (panel 5). This
BSM-mediated phagocytic activity was almost completely inhibited by A77
F(ab')2 (panel 6). Furthermore, a
mixture of uncoupled A77 F(ab')2 and 520C9
F(ab')2 could not enhance phagocytosis (panel
4), indicating the need for a conjugated A77 F(ab') x
520C9 F(ab') BSM to target tumor cells to effector cells leading to
activation of the MDM.
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B, optical sections encompassing the central portion of
dual-positive cells show the green CD14 fluorescence (from MDM)
appearing as a ring surrounding the red PKH-26-stained SKBR-3 cell
fragments. In a random analysis of 476 cells, 98% of dual-positive
cells showed the presence of SKBR-3 cell fragments inside the MDM. No
MDM/SKBR-3 cell conjugates were observed. Also, no intact SKBR-3 cells
were observed inside MDM. Figure 7 shows
that the BSM-mediated phagocytic activity was dose dependent and
saturated (nearly complete loss of tumor cells) at 0.1 µg/ml of the
BSM. A77 F(ab')2 almost completely blocked this
BSM-mediated phagocytosis.
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RI modulation
The modulation of FcRI expression upon BSM or A77 mAb binding
to monocytes or PMN was examined by flow cytometry. Figure 8 shows that 1 and 10 µg/ml whole A77
mAbs induced a 40 to 50% reduction of FcRI on PMN and monocytes
after overnight incubation at 37°C. This modulation activity did not
require cross-linking of the bound A77 by an anti-murine Ab.
However, 1 and 10 µg/ml of A77 x 520C9 BSM or A77
F(ab')2 induced little or no modulation of FcRI
under similar conditions, indicating that the Fc region of the A77 mAb
may be required to down-modulate FcRI expression.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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RI (CD89) can be exploited to develop effective immunotherapies.
The ability of FcRI to mediate phagocytosis, superoxide generation,
and induction of cytokine secretion has been described before (15, 16, 17, 18).
This report extends these observations to demonstrate the role of
FcRI in mediating ADCC of tumor targets by effector cells in the
whole blood or by freshly purified PMN or monocytes. The
FcRI-mediated cytolytic activity was demonstrated using two BSM
targeted to two distinct, well-characterized tumor-associated Ags
(HER2/neu and EGF-R) expressed on two different tumor cell
lines (SKBR-3 and A431). These BSM also mediated lysis of additional
tumor cell lines overexpressing EGF-R or HER2/neu. In whole
blood ADCC assays, A77 x H425 BSM-dependent lysis (40–50%) of
both HN5 (head and neck carcinoma line) and MDA-MB468 (breast carcinoma
line), which express levels of EGF-R comparable with A431 (skin
carcinoma line), was observed. Similarly, A77 x 520C9
BSM-mediated lysis of another HER2/neu overexpressing tumor
line, SKOV-3 (ovarian carcinoma line), in a whole blood ADCC assay.
These results suggest that FcRI-directed BSM may be broadly
applicable in a variety of malignancies. In addition, the results
reported here demonstrate that the FcRI-directed BSM also mediate
phagocytosis of HER2/neu-expressing tumor cells by MDM.
Thus, the FcRI-directed BSM could promote tumor cell destruction via
the effector cells circulating in peripheral blood (PMN and monocytes)
as well as the tissue macrophages (including tumor-associated
macrophages) without exogenous cytokine activation.
PMN are prolific effector cells involved in the defense against
invading infectious agents and at sites of inflammation. In addition to
FcRI, two known IgG-FcR (the transmembrane FcRIIa and
glycosylphosphatidylinositol-linked FcRIIIb) are constitutively
expressed on naive PMN. PMN can be stimulated in vivo with
granulocyte-CSF (G-CSF), IFN-, or granulocyte macrophage-CSF and in
vitro with IFN- to up-regulate expression of FcRI (33). Valerius
et al. (34) and Stockmeyer et al. (35) have reported that
FcR-directed BSM could mediate ADCC of tumor cells by PMN from
G-CSF-treated individuals. In this report, we demonstrate that
FcRI-directed BSM can promote the specific destruction of tumor
cells with purified PMN or whole blood (from volunteers without
cytokine treatments) as effector cells. Stockmeyer et al. (3 5)
have reported that when whole blood was used as a source of effector
cells, very limited Ab-dependent lysis (10%) of 520C9 Ab-coated
SKBR-3 cells was observed. We have also confirmed that 520C9 could not
mediate ADCC of SKBR-3 cells in whole blood (data not shown). These
results indicate that murine mAbs of IgG class may not be effective in
mediating ADCC of target cells under physiologic conditions. Results
presented in this report show that the BSM directed to FcRI could
mediate such ADCC activity with whole blood as effector cells in the
absence of presensitization of tumor cells by the BSM. This is relevant
for solid tumor therapy because if presensitization of tumor cells by
mAb/BSM is a prerequisite for tumor cell lysis in vivo, the tumor cell
destruction could be an inefficient process since a very small portion
of i.v.-administered mAb penetrates solid tumors (36). However,
i.v.-administered mAb/BSM immediately encounter FcR-expressing effector
cells in peripheral blood, of which PMN constitute the largest
population. The FcRI-directed BSM could coat effector cells in
peripheral blood, as shown for FcR-directed BSM (37), and could
engage in the destruction of tumor cells in circulation or upon
penetration into solid tumors that have not been presensitized.
Furthermore, this ADCC activity of FcRI-directed BSM may not be
dependent upon engagement of the IgA binding domain of FcRI, since
both the BSM used in our studies were directed to an epitope outside
the Fc binding domain of FcRI. It is expected that the FcRI on
effector cells in whole blood is saturated with serum IgA. It is not
known whether this receptor saturation plays a role in functional
activation of FcRI. On the other hand, the potent ADCC activity
mediated by these BSM in whole blood ADCC assays shows that saturation
of the Fc binding domain of the FcRI by serum IgA does not block
BSM-mediated ADCC activity under physiologic conditions. In addition to
the ADCC activity, some preliminary data indicate that PMN may also
participate in BSM-mediated FcRI-specific phagocytosis of target
cells (data not shown). Collectively, these results suggest that
FcRI is an important trigger receptor on PMN, and that BSM directed
to FcRI can be used to specifically recruit the cytotoxic potential
of PMN without exogenous activation with cytokines.
FcRI-directed BSM engage not only the cytotoxic activity of PMN but
also that of monocytes. A77 x 520C9 and A77 x H425 BSM
could mediate ADCC of either HER2/neu- or EGF-R-expressing
tumor cells by freshly purified monocytes from normal volunteers. Again
the FcRI-mediated cytolytic activity was not tumor Ag restricted.
The low level of BSM-independent (10%) lysis in all the experiments
described above indicates that freshly purified or whole blood PMN and
monocytes are incapable of inducing tumor cell lysis without activation
via a trigger receptor. Almost complete inhibition of BSM-directed
tumor cell lysis by A77 F(ab')2 suggests that these BSM
mediated the ADCC activity only via FcRI.
In addition to promoting extracellular lysis, the FcRI-directed BSM
mediated potent phagocytic activity. In particular, when MDM were used
as effector cells nearly 100% of tumor cells were phagocytosed. It is
likely that the BSM mediated both ADCC and phagocytic activities
simultaneously, and entire cells or fragments of lysed cells were
phagocytosed by the MDM via FcRI. These two mechanisms have been
shown to occur in concert for FcRI-mediated phagocytosis (32).
Confocal microscopy confirmed that the tumor cells are actually
ingested by MDM. It has been shown that Ag-presenting phagocytic cells
(such as MDM) can present Ags via both class I and class II pathways
after ingestion of Ag-bearing particles (38). Thus, the potent
phagocytosis of tumor cells via FcRI may lead to activation of both
humoral and cellular immune functions specifically directed to the
tumor-associated Ags.
These cytotoxic activities of FcRI-directed BSM are of therapeutic
value, because FcRI expression is limited primarily to only those
immune effector cells (PMN, monocytes, and macrophages) that
demonstrated the BSM-dependent cytotoxic activities described in this
report (4). On the other hand, FcR for IgG (FcR), the isotype of
almost all of the tumor-specific mAbs explored to date, are expressed
on noncytotoxic effector cells (e.g., FcRII on B cells and
platelets) or do not trigger cytotoxic functions on certain cytotoxic
effector cells (e.g., the glycosylphosphatidylinositol-linked
FcRIIIb on PMN) (33). Therefore, mAbs of IgG class may have limited
cell-mediated cytotoxic potential in vivo. This is indirectly suggested
by the very limited tumor cell lysis by 520C9 mAb (murine IgG1) in the
whole blood ADCC assay (35). On the other hand, because of its
distribution only on cytotoxic effector cells and its potent triggering
activity, FcRI appears to be an attractive candidate for
cell-mediated immunotherapies. Furthermore, the approach described here
can be employed to prepare FcRI-directed BSM using the existing high
affinity tumor-specific IgG mAbs. This will obviate the need to
generate new IgA class tumor-specific mAbs to exploit the cytotoxic
potential of FcRI.
Several BSM that engage FcRI to trigger effector cell functions have
been described (19, 22, 24, 31, 32, 34, 35). Included in these are BSM
that direct HER2/neu- or EGF-R-expressing tumor cells to
FcRI (22, 34, 35). FcRI, like FcRI, is found primarily on
cytotoxic myeloid effector cells. However, unlike the results reported
here for targeting FcRI, to achieve efficient PMN-mediated killing
through FcRI the PMN must be preactivated by cytokines. Valerius et
al. (34) and Stockmeyer et al. (35) have shown that PMN isolated from
G-CSF-treated individuals mediated potent ADCC through FcRI, whereas
PMN isolated from untreated individuals did not. Furthermore, when
whole blood from G-CSF-treated subjects was used as a source of
effector cells, significant ADCC using FcRI-directed bispecific Ab
was observed; little, if any, FcRI-mediated ADCC was observed using
whole blood from untreated donors (35). Interestingly, PMN in whole
blood from G-CSF-treated patients appear to be significantly less
efficient in FcRI-mediated tumor cell lysis, although the total
lysis is substantially enhanced because of the dramatic rise in the PMN
population after G-CSF treatment (39). Keler et al. (31) demonstrated
that although untreated monocytes could mediate FcRI-directed ADCC,
this activity was enhanced by treating the monocytes with IFN- in
vitro. Similarly, PMN isolated from normal volunteers required
treatment in vitro with IFN- to express FcRI and to mediate ADCC
through this receptor (31). In contrast to these observations, the data
presented here show that ADCC mediated through FcRI required no
prior activation of the effector cells. Studies are ongoing to
determine whether the already potent cytotoxicity mediated via FcRI
can be enhanced even further by cytokine treatment of effector
cells.
Finally, BSM binding to monocytes and PMN did not lead to
cross-linking and subsequent down-modulation of FcRI in the absence
of target Ag or target cells (Fig. 8). Therefore, the FcRI-directed
BSM can be used to "arm" the effector cells without activation by
receptor cross-linking, thus avoiding undesired systemic side effects.
These BSM-armed effectors are expected to be activated locally only
upon cross-linking of FcRI by tumor cells expressing appropriate
tumor Ag. Similar arming of monocytes by FcRI-directed BSM has been
shown (37); however, in this case pretreatment with G-CSF or IFN- is
required to engage the PMN effector population in vivo (40, 41). The
FcRI-directed BSM described here could engage monocytes, PMN, and
macrophages without cytokine pretreatment. In addition to the data
presented here, FcRI-directed BSM could be used to combat infectious
diseases, since most infectious agents (bacteria, viruses, fungi, etc.)
express unique Ags on their surface and several pathogen-specific Abs
have been described. This technology would be applicable in combating
antibiotic-resistant pathogens such as methicillin-resistant
Staphylococcus aureus or Candida spp., which
cause deleterious infections in T cell-deficient immunocompromised
patients. Intriguing results demonstrating phagocytosis of fungal
targets by FcRI-directed BSM have been described elsewhere (39). In
conclusion, the potent and specific cytotoxic activity of
FcRI-directed BSM described in this report point to a versatile
technology for the development of novel immunotherapies to combat
various malignancies and other disease conditions.
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Acknowledgments |
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Footnotes |
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2 Abbreviations used in this paper: FcRI, FcR for IgA; EGF-R, epidermal growth factor receptor; PMN, polymorphonuclear leukocytes; ADCC, Ab-dependent cell-mediated cytotoxicity; MDM, monocyte-derived macrophages; FcRI, FcR for IgG; o-PDM, ortho-phenylenedimaleimide; PE, phycoerythrin; G-CSF, granulocyte-CSF; BSM, bispecific molecules.
Received for publication August 15, 1997. Accepted for publication October 29, 1997.
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