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-Receptor I (CD89) Recruits Neutrophils as Effector Cells for CD20-Directed Antibody Therapy1
*
Division of Hematology/Oncology, Department of Medicine III, University of Erlangen-Nürnberg, Erlangen, Germany;
Medarex, Annandale, NJ;
Tenovus Research Laboratory, Southampton, U.K.; and
Department of Immunology and Medarex Europe BV, University Hospital Utrecht, Utrecht, The Netherlands
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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RI) and CD20, a broad range of B cell lines were effectively
killed. FcRI is expressed on monocytes/macrophages, neutrophils, and
eosinophils. As the numbers of these effector cells and their
functional activity can be enhanced by application of G-CSF or GM-CSF,
lysis via (FcRI x CD20) BsAb was significantly enhanced in
blood from patients during therapy with these myeloid growth factors.
Interestingly, the major effector cell population for this BsAb were
polymorphonuclear neutrophils, which proved ineffective in killing
malignant B cells with murine, chimeric IgG1, or Fc
RI- or
FcRIII-directed BsAbs against CD20. Experiments with blood from
human FcRI/FcRI double-transgenic mice showed corresponding
results, allowing the establishment of relevant syngenic animal models
in these mice. In conclusion, the combination of myeloid growth factors
and an (FcRI x CD20) BsAb may represent a promising approach
to improve effector cell recruitment for CD20-directed lymphoma
therapy. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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85% are of B cell origin, and most patients with
low or intermediate grade lymphomas, or relapses of high grade
lymphomas, have a poor prognosis despite major advances in chemo- and
radiotherapy, including bone marrow transplantation (2).
Since the description of the hybridoma technology by Köhler and
Milstein (3), more than two decades passed before the
mouse/human chimeric CD20 Ab C2B8 was approved by the Food and Drug
Administration as the first mAb for treatment in oncology. The CD20 Ag
seemed to be a particularly promising target for immunotherapy of B
cell neoplasms (4) because it is expressed on the cell
surface of >90% of malignant B cells but not on hemopoietic stem
cells, normal plasma cells, myeloid, T lineage, endothelial, or other
nonlymphoid cells (5). Upon binding of Abs, CD20 does not
significantly modulate or shed, and a plethora of potential effector
mechanisms of mAbs were shown to be recruited, such as Ab-dependent
cell-mediated cytotoxicity
(ADCC)3 by mononuclear
effector cells, complement-dependent lysis, initiation of intracellular
signals such as calcium fluxes, inhibition of cell growth, and
induction of cell differentiation. Importantly, CD20 Abs were shown to
induce apoptosis of malignant B cell lines, especially after intensive
cross-linking, e.g., by receptors for the Fc domain of IgG
(FcR)-expressing cells (6).
To recruit cell-mediated effector mechanisms, Abs must interact with Ig
FcRs, which are divided into Fc-, Fc
-, or FcRs, depending on
their specificity for IgA, IgE, or IgG, respectively (7).
FcR isoforms are grouped into two classes of low affinity receptors
named FcRII (CD32) and FcRIII (CD16) and a single high affinity
class, FcRI (CD64) (8). A pivotal role for FcRs as
mediators of therapeutic Ab effects in vivo was suggested by studies in
mice in which the signaling machinery of FcRs was disrupted by gene
targeting of the FcR common -chain (9). In contrast to
their littermates, these genetically modified animals were no longer
protected from tumor growth by therapeutic mAbs. Among other potential
FcR-mediated functions for therapeutic Abs, such as phagocytosis of
tumor cells (10) and, subsequently, enhanced presentation
of tumor Ags to T cells (11) or improved induction of
apoptosis by target Ag cross-linking (6), ADCC is
considered important in vivo (12). The capacity to mediate
ADCC has been demonstrated in vitro for monocytes/macrophages, NK
cells, as well as eosinophilic and neutrophilic granulocytes.
Neutrophils are increasingly recognized as an important effector cell
population for growth arrest and rejection of malignant tumors in vivo
(13). In vitro, polymorphonuclear neutrophils (PMNs) were
the predominant effector cell population for the killing of breast
cancer cells in the presence of HER-2/neu Abs, especially after
preactivation of neutrophils by G-CSF (14), which is known
to induce expression of FcRI as an additional cytotoxic trigger
molecule (15, 16). However, when we analyzed the capacity
of neutrophils to kill malignant B cells, we observed that they were
very efficient in killing B cells with Abs directed against HLA class
II but proved completely ineffective with Abs to other B
cell-associated Ags such as CD19, CD20, CD21, CD37, or CD38 (17, 18).
Bispecific Abs (BsAbs), containing one specificity against a tumor
target Ag and another specificity against select epitopes of an
activating FcR on cytotoxic cells, are an elegant way to improve
effector cell recruitment for Ab therapy (19, 20).
Recently, we demonstrated that, in addition to the IgG receptors
FcRI (CD64) and FcRIII (CD16), the myeloid receptor for IgA
(FcRI, CD89) is an interesting trigger molecule for BsAb therapy
(21). FcRI is constitutively expressed on
monocytes/macrophages, eosinophils, neutrophils, and some types of
dendritic cells, but importantly it is not found on noneffector cell
populations (22). Activation of FcRI was shown to
trigger phagocytosis, respiratory burst, cytokine release, and ADCC. As
some otherwise resistant solid tumor cell lines were effectively killed
by growth factor-primed PMNs in the presence of FcRI-directed BsAbs,
we were interested to test whether the Ag restriction of neutrophils in
killing malignant B cells could be overcome by targeting FcRI
instead of FcRs. As described in this manuscript, PMNs were indeed
found to effectively lyse malignant B cells with an (FcRI x
CD20) BsAb, but were again unable to kill CD20-positive B cells with
IgG or FcR-directed BsAbs against the CD20 target Ag. These results
demonstrate for the first time that the combination of myeloid growth
factors and (FcRI x CD20) BsAb may significantly improve
effector cell recruitment for CD20-directed immunotherapy.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Experiments reported here were approved by the Ethical Committee
of the University of Erlangen-Nürnberg (Erlangen, Germany), in
accordance with the Declaration of Helsinki. After informed consent,
10–20 ml of peripheral blood was drawn from healthy volunteers or from
patients receiving rhG-CSF (3–5 µg/kg of body weight, Neupogen;
Hoffmann-LaRoche, Basel, Switzerland) or rhGM-CSF (5 µg/kg of body
weight, Leukomax; Essex Pharma, Munich, Germany) based on clinical
indications. For analysis during growth factor treatment, patients had
at least 3 days of cytokine therapy. Relative fluorescence intensities
(RFIs) for expression of FcRI (CD89) or FcRI (CD64) on PMNs from
healthy donors or from patients treated with G-CSF or GM-CSF are
presented in Table I
. Staining for
FcRI was significantly higher than that for FcRI on healthy
donor- and GM-CSF-primed PMNs, but not on G-CSF-primed PMNs. Expression
of FcRI was significantly higher on G-CSF-primed PMNs compared with
healthy donor- or GM-CSF-primed neutrophils.
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RI/FcRI double-transgenic mice
FcRI/FcRI double-transgenic mice were generated by
crossing human FcRI- with human FcRI-transgenic mice. Human
FcRI-transgenic mice were generated by injection of an 18-kb human
genomic DNA fragment carrying the FcRIA gene
into FVB/N oocytes (23). A 41-kb cosmid clone containing
the human FcRI gene was used as a construct to
generate FcRI transgenic mice (24). Expression of
transgenes was checked by flow cytometry of peripheral blood cells,
using FITC-labeled anti-FcRI mAb 22 or anti-FcRI mAb A77
(both obtained from Medarex, Annendale, NJ), respectively. All mice
were bred at the Transgenic Mouse Facility of Utrecht University
(Utrecht, The Netherlands). To induce neutrophil FcRI expression and
to increase blood neutrophil counts, mice were s.c. injected with
murine G-CSF for 4 days (1.6 µg/mouse/day) before ADCC experiments.
Murine G-CSF was provided by Dr. J. Andresen (Amgen, Thousand
Oaks, CA).
Cell lines
The malignant B cell lines B acute lymphoblastic leukemia (BALL), RAJI (Burkitt’s lymphoma), ARH-77, and CESS (both mature B cell lines) were obtained from the American Type Culture Collection (Manassas, VA). RM-1 (EBV-transformed B cell line) and BJAB (Burkitt’s lymphoma) were obtained from Dr. G. Bonnard (National Cancer Institute, Bethesda, MD) and Dr. W. Leibold (Department of Veterinary Medicine, University of Hannover, Hannover, Germany), respectively. All cells were kept in RF10+ medium consisting of RPMI 1640 (Life Technologies, Paisley, U.K.) supplemented with 10% heat-inactivated FCS, 100 U/ml penicillin, 100 U/ml streptomycin, and 4 mmol/L L-glutamine (all obtained from Life Technologies).
mAbs and Ab constructs
Abs F3.3 (HLA class II, mIgG1), AT80 (CD20, mIgG1), and H147
(CD20, mIgG3) were generated, and WR17 (CD37, mIgG1; Ref.
25) and 1F5 (CD20, mIgG2a; Ref. 26) were
produced from the original hybridomas at the Tenovus Research
Laboratory (University of Southampton, Southampton, U.K.). CD20 Ab
MEM-97 (mIgG1) was provided by Dr. V. Horejsi (Institute for Molecular
Genetics, Prague, Czech Republic). B1 (mIgG2a), Leu-16 (mIgG1), and
mouse/human chimeric C2B8 (hIgG1) were purchased from Coulter (Hialeah,
FL), Dianova (Hamburg, Germany), and Hoffmann-LaRoche, respectively.
Abs A77 (FcRI, CD89; mIgG1), 22 (FcRI, CD64; mIgG1), 3G8
(FcRIII, CD16; mIgG1), 4G7 (CD19, mIgG1), FITC-labeled A77 or 22,
and 14.1 (a novel fully human IgG1 Ab against CD89) were obtained from
Medarex. PE-labeled Gr-1 Ab was obtained from PharMingen (San
Diego, CA).
BsAbs (FcRI x CD19), (FcRI x CD20), (FcRI x
CD37), (FcRI x HLA class II), (FcRI x CD20), and
(FcRIII x CD20) were produced by chemically cross-linking
F(ab') fragments of target Ag Ab 4G7 (CD19), 1F5 (CD20), WR17 (CD37),
or F3.3 (HLA class II) with trigger molecule Ab A77 (FcRI, CD89), 22
(FcRI, CD64), or 3G8 (FcRIII, CD16) as described
(27). Additional
(FcRI x CD20) BsAbs were generated using CD89 Abs A77 or 14.1,
and CD20 Abs AT80 or C2B8. Briefly, F(ab')2
fragments were produced by limited proteolysis with pepsin and were
then reduced with mercaptoethanol amine to provide Fab' with free
hinge-region sulfhydryl (SH) groups. The SH groups on one of the
Fab'(SH) partners were then fully alkylated with excess
o-phenylenedimaleimide (o-PDM) to provide free
maleimide groups (mal). Finally, the two preparations Fab'(mal) and
Fab'(SH) were combined at a ratio of 1:1 to generate heterodimeric
constructs. After purification by size exclusion chromatography and
characterization by HPLC, samples were sterilized by filtration and
stored at 4°C.
The chimeric Fab(Fc)2 construct of CD20 mAb 1F5
(ch1F5), consisting of a single Fab' fragment from mouse Ab 1F5
chemically conjugated to two human Fc fragments, was prepared as
reported (28). Briefly, F(ab')-o-PDM of 1F5
were produced as described above. To prepare human Fc, human serum
IgG was digested with papain, and resulting Fc fragments were
separated and purified. Following reduction of Fc fragments,
fragments were incubated with F(ab')-o-PDM to yield
Fab(Fc)2 constructs with mainly human IgG1 Fc
fragments.
Serial dilutions of Ab derivatives were analyzed for binding to
effector and target cells by indirect immunofluorescence. Half-maximal
binding to tumor cells occurred at 0.06, 0.05, and 0.09 µM for the
three ((22 x 1F5), (3G8 x 1F5), or (A77 x 1F5))
BsAbs, respectively, and at 0.5 µM for the chimeric 1F5 Ab.
Similarly, half-maximal binding to effector cells was determined at
0.012, 0.1, and 0.025 µM for the three BsAbs, respectively. Avidity
of parental mAbs 22 (FcRI) and A77 (FcRI) to isolated PMNs from
G-CSF-treated patients was 0.6 and 10 nM, respectively.
Isolation of mononuclear cells (MNCs) and neutrophil effector cells
Neutrophils were isolated by a method slightly modified from that described in (15). Briefly, citrate-anticoagulated blood was layered over a discontinuous Percoll (Seromed, Berlin, Germany) gradient consisting of 70 and 62% Percoll. After centrifugation, neutrophils were collected at the interphase between the two Percoll layers, and MNCs from the serum/Percoll interphase. Remaining erythrocytes were removed by hypotonic lysis. Purity of neutrophils was determined by cytospin preparations and exceeded 95%, with few contaminating eosinophils in healthy donors and G-CSF-treated patients and up to 25% eosinophils in preparations from GM-CSF-treated patients. Viability of cells tested by trypan blue exclusion was >95%. MNC contamination was <1% in all preparations.
Immunofluorescence analysis
For indirect immunofluorescence, polyclonal human IgG (4 mg/ml)
was added to inhibit nonspecific binding to FcRI. Cells were washed
three times in PBS supplemented with 1% BSA. FITC-labeled
F(ab')2 fragments of goat anti-mouse or
anti-human mAbs were used for staining. Cells were washed again and
analyzed on a flow cytometer (EPICS Profile; Coulter). For each cell
population, RFI was calculated as the ratio of mean linear fluorescence
intensity of relevant to irrelevant, isotype-controlled Abs.
ADCC assays
ADCC assays were performed as described (14). Briefly, target cells were labeled with 200 µCi 51Cr for 2 h. After extensive washing with RF10+, cells were adjusted to 105/ml. Whole blood or isolated effector cells (50 µl), sensitizing Abs, and RF10+ were added to round-bottom microtiter plates. Assays were started by adding the target cell suspension (50 µl), giving a final volume of 200 µl, and an E:T cell ratio of 40:1 with isolated human effector cells. After 3 hours at 37°C, assays were stopped by centrifugation, and 51Cr release from triplicate samples was measured in cpm. Percentage of cellular cytotoxicity was calculated using the formula: % specific lysis = (experimental cpm - basal cpm)/(maximal cpm - basal cpm) x 100, with maximal 51Cr release determined by adding perchloric acid (3% final concentration) to target cells, and basal release was measured in the absence of sensitizing Abs and effector cells. Ab-independent cytotoxicity (effectors without target Abs) was observed in whole blood assays and with mononuclear effector cells, but not with PMNs. ADCC experiments with murine whole blood were performed in duplicate.
Calcium mobilization assay
Intracellular free calcium levels were analyzed by a flow
cytometry assay (29). Whole blood from G-CSF-treated human
FcRI/FcRI double-transgenic mice was incubated with 0.2 x
PBS for 1 min to lyse erythrocytes. White blood cells were then
incubated with seminapthorhodafluor (SNARF)-1 (2.8 µM) and Fluo-3
(1.4 µM) (Molecular Probes, Eugene, OR) for 30 min at 37°C. After
washing, cells were incubated with anti-CD64 mAb 22 (10 µg/ml) or
anti-CD89 mAb A77 (10 µg/ml) for 30 min at room temperature,
washed twice, and resuspended in calcium mobilization buffer at a
concentration of 1 x 107 cells/ml. PMNs
were identified by forward and side scatter profiles, and cells were
measured at a rate of 
140 cells/s. The first 24 s of each run
were used to establish baseline intracellular calcium levels, after
which cross-linking goat anti-mouse IgG1 (Southern Biotechnology
Associates, Birmingham, AL) was added at concentrations ranging from 1
to 10 µg/ml. [Ca2+]i
baseline levels were subtracted from all measurements, and %
Fluo-3/SNARF-1 ratio was calculated by dividing the Fluo-3/SNARF-1
ratio at a given time point by the maximal Fluo-3/SNARF-1 ratio of each
individual experiment x 100%.
Statistical analysis
Group data are reported as mean ± SEM. Differences between groups were analyzed by unpaired (or, when appropriate, paired) Student’s t tests. Significance was accepted when p < 0.05.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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RI-directed BsAbs
against CD20
Chimeric IgG1 Abs against CD20 are particularly effective in
follicular or post transplant lymphoma patients, whereas results in
patients with other histologies, such as mantle cell lymphoma or
diffuse large cell lymphoma, are less impressive (30). We
established whole blood cytotoxicity assays against two prototypic
CD20-positive B cell lines: RAJI, a Burkitt’s lymphoma, which proved
sensitive for anti-CD20-mediated killing, and ARH-77, a mature B
cell line, which was not lysed by the chimeric CD20 Ab 1F5 (Fig. 1). Effector mechanisms operative in the
killing of RAJI cells were analyzed by fractionating whole blood into
plasma, MNCs, and granulocytes. Thus, we found chimeric Ab-mediated
killing of RAJI cells by plasma (43 ± 12%) and MNCs (45 ±
16%) but not by granulocytes (5 ± 5%, n = 4),
the latter in agreement with our previous observation that neutrophils
do not lyse malignant B cells in the presence of Abs to "classical"
B cell Ags (17, 18). Plasma-mediated lysis of RAJI cells
by chimeric 1F5 was completely abolished by heat inactivation of plasma
(56°C for 30 min), suggesting that complement-dependent cytotoxicity
was the underlying mechanism. As expected, ARH-77 cells were not killed
by any of these fractions in the presence of the chimeric CD20 Ab.
However, interestingly, both cell lines were effectively lysed by whole
blood from GM-CSF-treated patients using an (FcRI x CD20) BsAb
(Fig. 1). Analysis of the lytic fraction in whole blood identified PMNs
as the major effector population for this BsAb (68 ± 8, 16
± 3, and 3 ± 1% specific lysis for PMNs, MNCs, or plasma,
respectively; n = 5).
|
R-directed BsAbs with
(FcRI x CD20) BsAb
In previous experiments, we found neutrophils to mediate ADCC with
mouse/human chimeric IgG1 Abs, but especially G-CSF-primed neutrophils
were less effective with this isotype than, e.g., with murine IgG2a or
with FcRI-directed BsAbs (18). As recent results in
solid tumor models showed the most potent triggering of PMN-mediated
cytotoxicity with FcRI-directed BsAbs (21, 31), we
tested whether (FcRI x CD20) BsAb could overcome neutrophils’
target Ag restriction in killing malignant B cells. As demonstrated in
Fig. 2A, isolated PMNs from healthy donors proved to be
potent cytotoxic effector cells against ARH-77 cells with (FcRI
x CD20) BsAb, whereas the parental 1F5 (mIgG2a) Ab, the respective
chimeric human IgG1 construct, other murine CD20 Abs of different
isotypes as well as the clinically effective mouse/human chimeric Ab
C2B8 (hIgG1) were unable to recruit PMNs as cytotoxic effector cells.
This raised the question of whether FcRI- or FcRIII-directed
BsAbs could induce lysis of malignant B cells via the CD20 target Ag
using activated FcRI-expressing PMNs from G-CSF-primed patients. As
expected, these effector cells induced high levels of target cell
killing with (FcRI x HLA class II) BsAb (98 ± 2%
specific lysis at 2 µg/ml, n = 4). However, against
the CD20 target Ag, only the FcRI-directed BsAb proved effective
(Fig. 2B). To exclude that this cytotoxic activity was a
particular feature of the (A77 x 1F5) bispecific construct, other
(FcRI x CD20) derivatives were generated, including a novel
fully human CD89 Ab. As displayed in Fig. 2C, all four
bispecific constructs were similarly effective in mediating killing of
malignant B cells.
|
RI
x CD20) BsAb-mediated cytotoxicity
Lysis of the B cell lines BALL, BJAB, RAJI (both Burkitt’s
lymphoma), CESS, RM-1, and ARH-77 (all three mature B cell lines) was
compared using isolated PMNs from GM-CSF-treated patients in the
presence of murine Ab 1F5 (mIgG2a), mouse/human chimeric 1F5 (hIgG1),
or the respective (FcRI x CD20) BsAb (all at 2 µg/ml). All
these B cell lines strongly expressed the CD20 Ag (RFI ranging from
14.3 for ARH-77 to 41.4 for RAJI). With the FcRI-directed BsAb, PMNs
lysed all these B cell lines (specific lysis from 9 ± 4% for
BALL to 92 ± 5% for RAJI), whereas no killing was obtained with
the parental or the chimeric 1F5 Ab (Fig. 3). FcRI-mediated lysis of different B
cell lines did not correlate to their CD20 expression level.
|
RI x
CD20) BsAb
As neutrophil numbers and their functional capacity can be
enhanced by application of the myeloid growth factors G-CSF or GM-CSF,
lysis with (FcRI x CD20) BsAb was investigated with whole
blood from cytokine-treated patients or from healthy donors as effector
source. As expected, total leukocyte and PMN counts were significantly
higher in growth factor-treated patients compared with those in healthy
donors (19,900 ± 3,600/µl; 21,100 ± 2,400/µl;
6,700 ± 600 and 16,700 ± 3,200/µl; 13,800 ±
2,300/µl; 4,300 ± 600/µl for G-CSF, GM-CSF, and healthy
donors, n = 6, respectively). Importantly, cytotoxicity
in blood from cytokine-treated patients was significantly enhanced
compared with healthy donor blood, and occurred at 25-fold lower Ab
concentrations (Fig. 4). In these
experiments, no plasma-mediated lysis was observed, and cell-mediated
cytotoxicity resided predominantly in the numerically expanded PMN
fraction (data not shown). Interestingly, when analyzed at constant E:T
cell ratios, GM-CSF- but not G-CSF-primed PMNs were significantly more
effective with the FcRI-directed BsAb than healthy donor PMNs
(68 ± 8, 34 ± 7, and 32 ± 9%, n = 8,
respectively). Importantly, cytotoxicity by the chimeric CD20 Ab was
not enhanced in blood from growth factor-treated patients compared with
healthy individuals (11 ± 8, 5 ± 5, and 5 ± 2%
specific lysis in the presence of 2 µg/ml of chimeric 1F5 with whole
blood from healthy donors, G-CSF-, or GM-CSF-treated patients,
n = 4 triplets of donors, respectively).
|
RI- directed BsAbs
Experiments with mAbs and FcRI-directed BsAbs showed
neutrophils to mediate killing of malignant B cells only with HLA class
II-directed Abs (17, 18). Results with the (FcRI
x CD20) BsAb encouraged us to investigate other B cell-related Ags as
targets for FcRI-directed BsAbs. To obtain optimal activation of the
effector cell system, whole blood from GM-CSF-treated patients was used
for these experiments. Again, HLA class II proved to be the most
effective target Ag with high levels of killing occurring at very low
Ab concentrations (Fig. 5). However,
importantly, significant tumor cell lysis was now also observed with
(FcRI x CD20) and, to a limited extend, with (FcRI x
CD19) BsAbs, whereas the (FcRI x CD37) BsAb was not
effective.
|
RI- or
FcRI-directed BsAbs using blood from mice transgenic for human
FcRI and FcRI
Syngenic animal models may provide important information for
relevant details of clinical trials, provided these models closely
reflect the human situation. Blood from G-CSF-treated transgenic mice
expressing both human FcRI and human FcRI was analyzed as
effector source against ARH-77 malignant B cells using FcRI- or
FcRI-directed BsAbs against CD20. Like neutrophils from
G-CSF-treated patients, PMNs from these transgenic animals expressed
comparable levels of human FcRI and human FcRI (Fig. 6A). For control, blood from
transgenic animals mediated efficient lysis with (FcRI x HLA
class II) BsAb (32 ± 7%, n = 6). However, with
CD20 as target Ag, only the FcRI- but not the FcRI-directed
construct was effective (Fig. 6B). Looking for a possible
explanation for the superior capacity of the FcRI transgene to
trigger ADCC, the ability of FcRI and FcRI to initiate an early
signaling event was assessed. Cross-linking FcRI, expressed on
FcRI/FcRI double-transgenic PMNs, triggered a very rapid increase
in intracellular free calcium levels
([Ca2+]i). Even though
cross-linking of FcRI on the same cells led to increased
[Ca2+]i levels as well,
this rise was always delayed compared with FcRI (Fig. 7), irrespective of the concentration of
the cross-linking goat anti-mouse Ab.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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RI-directed BsAbs against
"classical" B cell Ags, including CD20 (17, 18). Here
we show that this Ag restriction in neutrophil-mediated lysis can be
overcome by targeting FcRI (CD89) instead of FcRI (CD64). The
high cytotoxic activity of (FcRI x CD20) BsAbs was confirmed
using four different derivatives, including the clinically effective
CD20 Ab (C2B8) and a novel fully human CD89 Ab (14.1). Together, there
is a clear hierarchy in lysis via different target molecules on tumor
cells (with HLA class II > CD20 > CD19 > CD37), as
well as in cytotoxic trigger molecules on effector cells (with
FcRI > FcRI), the latter more obvious with suboptimal
target Ags. Further complexity is introduced by different effects of
G-CSF and GM-CSF on FcR expression and function. Thus, G-CSF but not
GM-CSF induced expression of FcRI and enhanced lysis via
FcRI-directed BsAbs. In contrast, FcRI-mediated killing was
increased by both G-CSF and GM-CSF, although FcRI expression was not
increased by either cytokine. Increased killing was caused by higher
effector cell numbers and enhanced cytotoxicity per cell in the case of
GM-CSF, but was only due to increased numbers of effector cells (which
were not stimulated compared with healthy donor cells) in the case of
G-CSF.
Similar results as with human effector cells were obtained when
BsAb-mediated killing of B cells was analyzed with blood from
double-transgenic mice expressing both human FcRI and human FcRI,
indicating that these differences in the killing capacity were truly
trigger molecule dependent. Both FcRI (CD89) and FcRI (CD64)
belong to the family of multi-chain immune recognition receptors
(MIRR), in which ligand-specific -chains form receptor complexes
with shared immunoreceptor tyrosine-based activation motif-containing
molecules named 
-, -, or 
-chains in the case of FcRs. These
signaling molecules, of which the FcR -chain is most widely
expressed, couple these membrane receptors to the intracellular
signaling machinery of Src and Syk protein tyrosine kinases
(32). Interestingly, FcR -chain facilitates surface
expression and signaling of both FcRI and FcRI (33, 34), suggesting that both receptors activate similar
immunoreceptor tyrosine-based activation motif-dependent intracellular
signaling pathways (35). Reasons for the observed
differences between both FcRs in the successful generation of a
cytolytic cascade are not defined at the moment and were most striking
for G-CSF-primed PMNs, which expressed comparable levels of both
FcRI and FcRI. Potential explanations include 1) different
killing mechanisms of PMNs activated by FcRI- compared with
FcRI-directed BsAbs; 2) improved PMN activation via FcRI either
by recruitment of additional signaling pathways or by better
interaction between FcRI and the common FcR -chain
(33); and 3) different on- and off-rates of the FcRI-
relative to the FcRI-directed Ab, leading to qualitative differences
in FcR triggering (as has been documented for T cell receptor-mediated
cell activation; Ref. 36). Our observation that FcRI
cross-linking consistently leads to more rapid rises in
[Ca2+]i supports the idea
that FcRI triggers signaling pathways more efficiently than FcRI.
Interestingly, the difference in the time to maximal
[Ca2+]i levels was more
pronounced with lower concentrations of cross-linking Ab, suggesting
that FcRI may be a more potent trigger molecule in situations of
limited opsonization with BsAb. Moreover, FcRI was also found to be
more effective in the generation of an oxidative burst than Fc
receptors (37, 38). However, reactive oxygen products
appeared not involved in neutrophil-mediated lysis because PMNs
from patients with chronic granulomatous disease were not impaired in
killing malignant B cells (our unpublished observations). Generation of
novel reagents including FcRI- or FcRI-directed Abs with
different affinities, and chimeric Abs of human IgG and IgA isotypes
(as natural ligands for these receptors), may help to address some of
these questions.
At the moment, chimeric CD20 Abs are probably the best example that
unconjugated mAbs can constitute an additional treatment option in
oncology. However, clinical results vary and seem to depend, e.g., on
patients’ histological subtype, indicating that further improvements
of efficacy are needed (30, 39). In vivo, therapeutic Abs
compete with high levels of endogenous Igs for binding to FcR.
Furthermore, Fc receptors are also expressed on cells lacking
cytotoxic activity such as platelets or B cells, and some Fc
receptor isoforms expressed on cytotoxic cells (e.g., FcRIIb,
FcRIIIb) bind Igs, but do not trigger cytotoxicity, both potentially
scavenging therapeutic Ab. This manuscript describes that an
(FcRI x CD20) BsAb is more effective than the respective
mouse/human chimeric IgG1 construct in directly killing a broad range
of B cell lines, especially when target cells were more complement
resistant. Interestingly, PMN-mediated killing of B cells does not
appear to be a simple cross-linking phenomenon of target Ags, as
indicated by the lack of activity of the (FcRIII x CD20) and
(FcRI x CD20) derivatives. In addition, FcRI-mediated lysis
was significantly enhanced in blood from G-CSF- or GM-CSF-treated
patients. The reason for this enhanced killing during growth factor
therapy lies in the fact that FcRI-directed BsAbs, but not chimeric
CD20 Abs, recruited neutrophils as major cytotoxic cell population.
Generation of human FcRI (23), human FcRI
(24), and human FcRI/FcRI double-transgenic mice
(this manuscript) will help to establish relevant syngenic animal
models for the evaluation of BsAb approaches, as ex vivo results with
blood from these animals closely reflected the human situation. These
animal models are expected to provide relevant information on important
details for future clinical trials such as dosing of BsAbs and timing
of Ab and cytokine applications. A clinical phase I trial with an
(FcRI x CD20) BsAb in combination with GM-CSF is expected to
commence soon. Provided that these phase I data show an acceptable
toxicity profile as found in similar studies with FcRI-directed
BsAbs in solid-tumor patients (40, 41), this combination
may become a promising approach to enhance efficacy of CD20-directed
lymphoma therapy.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Bernhard Stockmeyer, Division of Hematology/Oncology, Department of Medicine III, University Erlangen-Nürnberg, Krankenhausstrae 12, 91054 Erlangen, Germany.
3 Abbreviations used in this paper: ADCC, Ab-dependent cell-mediated cytotoxicity; BsAb, bispecific Ab; FcRI, myeloid Fc receptor for IgA; FcR, receptors for the Fc domain of IgG; PMNs, polymorphonuclear neutrophils; MNC, mononuclear cell; RFI, relative fluorescence intensity; BALL, B acute lymphoblastic leukemia; SH, sulfhydryl; o-PDM, o-phenylenedimaleimide; SNARF, seminapthorhodafluor.
Received for publication January 19, 2000. Accepted for publication August 17, 2000.
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). Murine
white blood cells were labeled with Fluo-3 and SNARF-1. After
incubation with A77 or 22 to stain Fc