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of June 16, 2017.
Antibodies That Efficiently Form Hexamers
upon Antigen Binding Can Induce
Complement-Dependent Cytotoxicity under
Complement-Limiting Conditions
Erika M. Cook, Margaret A. Lindorfer, Hilma van der Horst,
Simone Oostindie, Frank J. Beurskens, Janine Schuurman,
Clive S. Zent, Richard Burack, Paul W. H. I. Parren and
Ronald P. Taylor
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Print ISSN: 0022-1767 Online ISSN: 1550-6606.
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J Immunol 2016; 197:1762-1775; Prepublished online 29
July 2016;
doi: 10.4049/jimmunol.1600648
http://www.jimmunol.org/content/197/5/1762
The Journal of Immunology
Antibodies That Efficiently Form Hexamers upon Antigen
Binding Can Induce Complement-Dependent Cytotoxicity
under Complement-Limiting Conditions
Erika M. Cook,* Margaret A. Lindorfer,* Hilma van der Horst,† Simone Oostindie,†
Frank J. Beurskens,† Janine Schuurman,† Clive S. Zent,‡ Richard Burack,x
Paul W. H. I. Parren,†,{ and Ronald P. Taylor*
A
nti-tumor mAbs that are used in the immunotherapy of
cancer can promote destruction of cancer cells by several
mechanisms (1–5). It is now well-recognized that amino
acid or carbohydrate changes engineered into the Ab Fc regions can
substantially enhance their cytotoxic action because of increased and
more effective use of immune-based effector functions (2, 6–11). For
example, our groups have recently reported that single amino acid
*Department of Biochemistry and Molecular Genetics, University of Virginia School
of Medicine, Charlottesville, VA 22908; †Genmab, 3584 CM Utrecht, the Netherlands; ‡Wilmot Cancer Institute, University of Rochester Medical Center, Rochester,
NY 14642; xDepartment of Pathology, University of Rochester Medical Center,
Rochester, NY 14642; and {Department of Immunohematology and Blood Transfusion, Leiden University Medical Center, 2333 ZA Leiden, the Netherlands
ORCIDs: 0000-0002-6383-1560 (E.M.C.); 0000-0003-4203-405X (M.A.L.); 0000-00027165-5771 (F.J.B.); 0000-0002-9738-9926 (J.S.); 0000-0001-6099-3313 (C.S.Z.); 00000002-4365-3859 (P.W.H.I.P.); 0000-0003-1578-8382 (R.P.T.).
Received for publication April 12, 2016. Accepted for publication June 28, 2016.
This work was supported by a grant from Genmab.
Address correspondence and reprint requests to Dr. Ronald P. Taylor, Department of
Biochemistry and Molecular Genetics, University of Virginia School of Medicine,
Charlottesville, VA 22908. E-mail address: [email protected]
The online version of this article contains supplemental material.
Abbreviations used in this article: Al, Alexa Fluor; ALM, alemtuzumab; BDSS,
bright detail similarity score; CDC, complement-dependent cytotoxicity; CLL,
chronic lymphocytic leukemia; dpl, depleted; fB, complement factor B; fD, complement factor D; GMI, geometric mean intensity; Hx, hexamer formation enhanced
mAb; IgM-7D8, IgM form of mAb 7D8; MAC, membrane attack complex; MESF,
molecules of equivalent soluble fluorochrome; NHS, normal human serum; OFA,
ofatumumab; RT, room temperature; RTX, rituximab; TMRM, tetramethylrhodamine
methyl ester; TP3, TO-PRO-3; UVA, University of Virginia.
This article is distributed under The American Association of Immunologists, Inc.,
Reuse Terms and Conditions for Author Choice articles.
Copyright Ó 2016 by The American Association of Immunologists, Inc. 0022-1767/16/$30.00
www.jimmunol.org/cgi/doi/10.4049/jimmunol.1600648
changes in the Fc region of CD20 and CD38 mAbs enhance their
ability to form hexamers upon binding to their cognate Ag expressed
on B cells, thus increasing C1q binding and initiating complement
activation, which is rapidly followed downstream by very high levels
of complement-dependent cytotoxicity (CDC) (8, 12). The complement cascade terminates with the insertion of multiple copies of the
membrane attack complex (MAC, C5b-9) which pierces the cell
membrane. A threshold level of MAC binding promotes plasma
membrane permeability and influx of water and ions that kills the
targeted cell (13–18). Increasing evidence indicates that under physiologic conditions, as a consequence of this permeabilization, the rapid
rise in the concentration of intracellular Ca2+ poisons the cell and is the
most proximate mediator of MAC-induced cell death (12, 19–22).
Upstream steps in the complement cascade, which include both C1q
binding and C3b deposition, also require threshold levels for cytotoxic
efficacy (23, 24); normal and malignant cells can use a variety of
defensive measures to inhibit or neutralize these upstream steps (25–
27). Moreover, nucleated cells can extrude or internalize the MAC,
and therefore, a sufficient number of copies of the cell-bound MAC
must be generated to overwhelm these defenses (13–15, 28–32). On
the basis of these considerations, an important question must focus on
how effectively a given immunotherapeutic mAb can make use of the
finite supply of complement in the circulation and in other compartments to ensure targeting and destruction of tumor cells. For example,
in mAb-based treatment of patients with chronic lymphocytic leukemia (CLL), it is quite possible to infuse sufficient CD20 mAb to
saturate all available binding sites on circulating malignant B cells.
However, under these conditions and at the high cell burdens common
in CLL, complement activation by the mAb-opsonized cells can exhaust complement, thus compromising additional mAb-based therapy
dependent on CDC for periods of days or weeks (24, 33–36).
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Recently, we demonstrated that IgG Abs can organize into ordered hexamers after binding their cognate Ags expressed on cell
surfaces. This process is dependent on Fc:Fc interactions, which promote C1q binding, the first step in classical pathway complement activation. We went on to engineer point mutations that stimulated IgG hexamer formation and complement-dependent cytotoxicity (CDC). The hexamer formation–enhanced (HexaBody) CD20 and CD38 mAbs support faster, more robust CDC than
their wild-type counterparts. To further investigate the CDC potential of these mAbs, we used flow cytometry, high-resolution
digital imaging, and four-color confocal microscopy to examine their activity against B cell lines and primary chronic lymphocytic
leukemia cells in sera depleted of single complement components. We also examined the CDC activity of alemtuzumab (antiCD52) and mAb W6/32 (anti-HLA), which bind at high density to cells and promote substantial complement activation. Although
we observed little CDC for mAb-opsonized cells reacted with sera depleted of early complement components, we were surprised to
discover that the Hexabody mAbs, as well as ALM and W6/32, were all quite effective at promoting CDC in sera depleted of
individual complement components C6 to C9. However, neutralization studies conducted with an anti-C9 mAb verified that C9 is
required for CDC activity against cell lines. These highly effective complement-activating mAbs efficiently focus activated
complement components on the cell, including C3b and C9, and promote CDC with a very low threshold of MAC binding, thus
providing additional insight into their enhanced efficacy in promoting CDC. The Journal of Immunology, 2016, 197: 1762–1775.
The Journal of Immunology
Engineered hexamer-formation enhanced CD20 mAbs (HexaBody
molecules) support faster, more robust CDC than their wild-type
counterparts (12). To further investigate the CDC potential of these
mAbs, we have examined their CDC activity against both B cell lines
and primary CLL cells, in sera depleted (dpl) of single complement
components, with a particular focus on use of and requirements for
C9, because of its key role in MAC-mediated cell killing.
Materials and Methods
Cells
B cell lines were cultured as described previously (24). CLL cells were
purified from blood of untreated de-identified CLL patients (University of
Rochester) in accordance with protocols of the University of Rochester
Institutional Review Board. Cells were stored frozen in liquid N2 and
thawed for 1 h at 37˚C immediately before use (12, 36).
Reagents
FIGURE 1. Z138 cells reacted with Hx7D8, IgG1-7D8, Hx-RTX, or W6/32 in NHS
or in C9-dpl sera are very rapidly killed by
CDC, but much more MAC is demonstrable
bound to cells reacted in complete NHS than
in C9-dpl sera. Less CDC is seen for cells
reacted with RTX. (A and B) The percentage
of CDC is defined by uptake of TP3. For
incubation times of either 90 or 900 s, in
NHS or C9-dpl sera, p , 0.001 for Hx-RTX
versus IgG1-RTX, and for Hx-7D8 versus
IgG1-7D8 (n = 8 in each case). (C and D)
MAC binding is determined by probing with
FITC mAb aE11, specific for a neoepitope of
C9 expressed in the MAC. (E and F) The
percentage of all cells that has an FITC mAb
aE11 signal above background. Most cells
reacted in C9-dpl sera score as weakly positive for C9. mAb Hx-b12 serves as a background control. Also see Supplemental Fig. 1.
H and L chain encoding plasmids, essentially as described by Vink et al. (39).
Alemtuzumab (ALM) was obtained from the University of Virginia hospital
pharmacy. Control hexamerization-enhanced mAb, IgG1 Hx-b12, HIVspecific (40), was used to determine background signals for mAb binding
and CDC. FITC-conjugated anti-C9 mAb aE11, specific for a C9 neoepitope
present in the membrane attack complex (41), was from Hycult. Neutralizing
mAb 22, specific for C9, was from Pierce. dpl sera and purified C9 were from
Complement Technologies. Tetramethylrhodamine methyl ester (TMRM) and
TO-PRO-3 (TP3) were from Life Technologies. mAb 3E7 (mouse IgG1),
specific for human C3b/iC3b, and mAb W6/32 (mouse IgG2a, fixes complement), specific for human HLA, have been described (33, 42–44) MAbs
were labeled with Alexa Fluor (Al) N-hydroxysuccinimide esters (Life
Technologies), according to the manufacturer’s instructions.
CDC and C3b and C9 deposition
Cells were mixed with mAb-supplemented normal human serum (NHS;
typically final 50 or 25% for NHS, final 25% for depleted sera because of
cost) and unless otherwise specified, incubated at 37˚C for various times.
Samples were quenched with ice-cold PBS, washed twice (800 3 g,
3 min), and probed with 10 mg/ml FITC mAb aE11 in 2 mg/ml mouse IgG,
30 min at room temperature. Cells were then washed once and stained with
TP3 to measure viability by flow cytometry (FACSCalibur). C3b deposition was determined as above, except cells were probed with 10 mg/ml
Al488 3E7, 30 min at room temperature. Mean fluorescent intensities were
converted to molecules of equivalent soluble fluorochrome (MESF) (33).
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IgG1 CD20 mAbs rituximab (RTX), ofatumumab (OFA), 7D8, and corresponding hexamerization-enhanced mAbs (Hx designates IgG1 molecules
containing an E430G mutation) were described previously (8, 12, 37, 38). The
IgM form of mAb 7D8 (IgM-7D8) was produced by transient expression in
Freestyle HEK293F cells (Thermo Fisher Scientific) by cotransfection of
1763
1764
Unreacted cells (time zero) were first quenched with cold PBS and then
combined with mAb and NHS and immediately centrifuged.
C9 neutralization
Cells were pelleted and resuspended to a final density of 5 3 106 cells/ml in
25% NHS or 25% C9-dpl sera containing varying amounts of the neutralizing anti-C9 mAb 22 or an IgG1 isotype control. The CDC reaction
was initiated by addition of the opsonizing mAb. After incubation at 37˚C
for 15 min, samples were washed twice and probed with FITC mAb aE11,
as described above, and then stained with TP3 to measure viability.
Binding assays and determination of CD20 levels
Z138 cells (5 3 106 cells/ml), in BSA/PBS, were incubated with varying
Al555 mAb inputs for 30 min at room temperature, quenched with ice-cold
BSA/PBS, and centrifuged at 800 3 g, 3 min, 4˚C, and then analyzed by
flow cytometry. CD20 levels on B cells from untreated CLL patients were
determined as previously described, with minor modifications (24): cells
were reacted with saturating amounts of Al488 Hx-7D8, washed, and
probed with Al647 mAb HB43 (anti-human Fc specific). The relative
levels of CD20 are expressed in MESF units based on the Al647 signal.
Scanning confocal microscopy
Multispectral fluorescence imaging
CDC and colocalization were analyzed with an Amnis Imagestream Mark II
as described previously (23, 45). For colocalization experiments, unlabeled
FIGURE 2. Sera depleted of one of several terminal
(but not upstream) classical pathway complement components support CDC mediated by several different
mAbs. (A) mAbs Hx-7D8 and ALM promote robust CDC
of CLL cells after reaction for 15 min in 25% NHS or
C9-dpl sera. The results are the average for duplicate
determinations on cells from eight different CLL patients
for Hx-7D8 and the average for cells from four of the
eight patients for ALM; means and SD are displayed. No
CDC is observed if Hx-7D8 or ALM is reacted with CLL
cells in C1q-dpl sera. Differences between C1q-dpl and
C9-dpl versus NHS are significant, as illustrated. (B and
C) Both Hx-7D8 and W6/32 mediate CDC of Z138 cells
in sera depleted of terminal pathway components or of
complement factor B (fB) or complement factor D (fD).
n = 4–6. Significant differences versus NHS are noted.
(D and E) Both Hx-7D8 and ALM mediate CDC of CLL
cells in sera depleted of terminal pathway components or
of fB or fD. The averaged results for duplicate determinations on cells from eight different CLL patients are
provided. The results for C1q dpl, C9-dpl, and NHS are
the same as in (A), and are repeated to allow for ready
inspection and comparison with the other depleted sera.
*p , 0.05, **p , 0.01, ***p , 0.001.
cells were reacted with Al-labeled mAb in 50% NHS for varying times at
37˚C. Samples were quenched with ice-cold BSA/PBS, washed, and probed with Al-labeled mAbs specific for C3b/iC3b (3E7) along with FITC or
Al594 anti-C9 (aE11). The cells were then washed again filtered, pelleted,
and resuspended in BSA/PBS at ∼4 3 107 cells/ml and then analyzed.
Alternatively, for CDC, cells were either used unlabeled or first stained
with TMRM and then reacted with unlabeled mAbs in NHS for varying
times and then similarly processed and probed. Cell killing was measured
by uptake of TP3. Images (5,000–10,000 cells/sample) were captured at
360 magnification.
Statistical analysis
All experiments were performed at least two or more times (see figure
legends). Error bars represent SDs for two or more replicates. Statistical
significance for experiments with cell lines was evaluated in unpaired single
tail t tests, based on equal variances, with the hypothesis that the Hx-mAbs
would enhance CDC. Experiments with B cells from three to eight CLL
patients were evaluated with paired t tests (SigmaPlot). The following
symbols were used to denote significant differences: *p , 0.05, **p ,
0.01, and ***p , 0.001.
Results
Demonstration of CDC of cells in C9-dpl sera
We first examined the kinetics of mAb-promoted, complementmediated killing of Z138 cells in both NHS and in C9-dpl sera:
quite effective and rapid killing was mediated by CD20 mAbs
IgG1-7D8, Hx-7D8, and Hx-RTX under both conditions (Fig. 1A,
1B). To test whether the high levels of CDC achieved in C9-dpl
sera are unique to CD20 mAbs, we also examined CDC mediated
by the HLA-specific mAb W6/32. Indeed, at the same concentration used for the CD20 mAbs (10 mg/ml), W6/32 also promoted
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B cells were stained with TMRM according to the manufacturer’s instructions and
mixed with NHS or C9-dpl sera containing Hx-7D8 or W6/32, Al405 3E7, FITC
aE11, and TP3 (final concentrations: 35% serum, 10 mg/ml mAb, 20 mg/ml
Al405 3E7, 10 mg/ml FITC aE11, and 10 mM TP3). Then, 5 ml of the cell
suspension (4 3 107 cells/ml) was applied to glass-bottom poly-D-lysine–treated
35-mm petri dishes (Mattek). Real-time images were captured at room temperature or at 37˚C with a Zeiss LSM 700. Movies were extracted using Zen software.
RAPID CDC OF NUCLEATED CELLS
The Journal of Immunology
very rapid and substantial CDC in both NHS and in C9-dpl sera.
We also found, consistent with several reports (23, 24, 46), that
IgG1-RTX gave less CDC and was more active in NHS than in
C9-dpl sera.
We also measured the kinetics of mAb-mediated MAC binding
by probing the cells with FITC mAb aE11, specific for the neoepitope expressed on C9 in the MAC (41). C9 binding is easily
demonstrable on cells reacted with the mAbs in NHS (Fig. 1C,
1E), and the amount of C9 bound to the cells appears to peak and
then drop at later times, likely because of disintegration of the
FIGURE 4. In both NHS and in C9-dpl sera, Hx-7D8 has
considerably more CDC activity at low concentrations than
IgG1-7D8 or W6/32. (A) Binding isotherms for Al555labeled mAbs reacted with Z138 cells in BSA-PBS. All
three mAbs had F/P ratios of ∼4.5. The x-axis is plotted
on a log scale, but the data gave a good fit in a binding
isotherm (see text). At saturation, considerably more W6/32
binds to the cells than do the CD20 mAbs, which bind
with higher avidity. (B and C) Dose-response CDC of
Z138 cells (n = 4). (D and E) Dose-response tests for
duplicate determinations of CDC of CLL cells from three
patients (averages and SD). In (B)–(E), differences in
CDC for cells reacted with Hx-7D8 versus IgG1-7D8 are
highly significant. **p , 0.01, ***p , 0.001.
cells. The amount of C9 bound to cells reacted with Hx-7D8 in
NHS for 60 s is .2-fold greater than the amount bound to cells
reacted with any of the other mAbs. Even though .90% CDC is
observed for Z138 cells reacted with Hx-7D8, IgG1-7D8, or W6/
32 in C9-dpl sera, the amount of C9 binding for these cells is only
modestly above background, and the percentage of cells that
register as C9 positive is reduced considerably (Fig. 1D, 1F).
Moreover, with respect to the magnitude of the C9 binding signal,
we find that even in intact NHS, cell killing tends to lead C9
binding. That is, at early times in the reaction (30–60 s), a substantial fraction of cells are killed when the amount of C9 deposited is relatively low. We conducted ELISAs to measure the
amount of C9 in C9-dpl sera (47), and we found that the C9
concentration was quite low, #250 ng/ml, which corresponds to
removal of .99% of the C9 from serum based on a normal value
of 60 mg/ml (48). That is, very small amounts of bound C9 appear
to be adequate to kill the cells.
Analyses based on dot plots (TP3 versus C9 binding; Supplemental
Fig. 1) reveal that when cells are reacted in NHS, few if any TP3positive/C9-negative cells can be identified, indicating that under
this condition all of the dead cells have taken up measureable
amounts of C9. However, in C9-dpl sera, we could identify a subpopulation of cells that appeared to be TP3 positive/C9 negative. On
the basis of these experiments alone, we cannot determine whether
absolutely no C9/MAC is bound to some of the dead cells or
whether the flow cytometry measurement is simply not sufficiently
sensitive to detect very small amounts of cell-bound C9.
ARH-77 cells are particularly resistant to CDC mediated by
CD20 mAbs (43), but as we have previously reported, they can be
killed by CDC upon reaction with mAb W6/32 (42), and in fact,
CDC mediated by mAb W6/32 is also demonstrable in C9-dpl sera
and is approximately comparable to CDC in NHS (Supplemental
Fig. 2). Once again, we find that there is very little binding of the
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FIGURE 3. Hx-7D8 promotes high levels of CDC on CLL B cells regardless of CD20 density. CLL B cells from six untreated patients were
subjected to CDC mediated by Hx-7D8, IgG1-7D8, or OFA. Hx-7D8 was
considerably more effective than the other two mAbs in promoting CDC
(n = 6, p , 0.01). Relative CD20 levels were determined as described in
Materials and Methods.
1765
1766
FITC anti-C9 mAb aE11 to ARH-77 cells killed by W6/32 in C9-dpl
sera, thus providing additional support for our observation that certain mAbs can mediate rapid and effective CDC in C9-dpl sera.
mAb-mediated CDC of primary CLL cells in C9-dpl sera
To evaluate the possible translational implications of our findings,
we investigated whether primary CLL cells could also be killed by
CDC in C9-dpl sera. Purified B cells from CLL patients were
reacted with either Hx-7D8 or with CD52-specific mAb ALM, a
mAb that is also particularly effective at promoting CDC (34, 49,
50). Substantial and roughly comparable levels of CDC are mediated by both Hx-7D8 and ALM in both intact NHS and in
C9-dpl sera (Fig. 2A). However, no cytotoxicity was observed in
C1q-dpl serum, which is consistent with our previous findings
(23, 24) that indicated that the classical pathway of complement
mediates CDC of cells opsonized with CD20 mAbs or with ALM.
Generalization of CDC to sera depleted of other complement
components
C9-dpl sera (Fig. 1). To better compare the CDC efficacy of the
mAbs, we next investigated whether lower concentrations of Hx-7D8,
compared with IgG1-7D8 and W6/32, could promote relatively more
CDC in either NHS or in C9-dpl sera (Fig. 4B, 4C). That is, the
question is whether small amounts of the cell-bound Hx-7D8 mAb
(far below saturation of binding) are better able to activate complement than comparable amounts of cell-bound IgG1-7D8 or W6/32.
Indeed, at 10-fold lower mAb concentrations (1 mg/ml), when approximately equal amounts of the mAbs are bound to the cells,
Hx-7D8 indeed mediates more CDC of Z138 cells, both in NHS and
in C9-dpl sera, than does IgG1-7D8 or W6/32.
We conducted similar dose-response experiments to examine
mAb-mediated CDC of CLL cells (Fig. 4D, 4E). The results again
indicate that, compared with IgG1-7D8, low concentrations of
Hx-7D8 promote considerably more CDC in both NHS and in
C9-dpl sera. Even at a concentration of 0.3 mg/ml, Hx-7D8 still
mediates some CDC of CLL cells in C9-dpl sera, whereas no killing
is observed for the IgG1-7D8 wild-type. W6/32 does not bind to
CLL cells and therefore in this case served as a negative control.
Almost all of the experiments we have presented were conducted
under conditions where the final cell concentration was 2.5–5 3
106 cells/ml. At higher cell concentrations, differences between
C9-dpl sera and intact NHS in supporting CDC were more pronounced: CDC of Z138 cells (5 3 107 cells/ml) mediated by Hx7D8 was 69% in 25% NHS but only 35% in 25% C9-dpl sera.
Potency of Hx-7D8 as a function of CD20 density
The B cells of CLL patients can vary considerably with respect to
expression of CD20, and it is well-established that higher levels of
CD20 on CLL B cells presage more effective CDC mediated by CD20
mAbs such as RTX or OFA (24, 51, 52). Therefore, we next compared Hx-7D8 with both IgG1-7D8 and OFA, with respect to their
abilities to mediate CDC of CLL cells from six different patients in
which there was an approximate 10-fold range of CD20 expression
levels. We find that for B cells of the patient with the highest CD20
level, all three mAbs are quite effective in mediating CDC (Fig. 3).
However, for CLL B cells with lower levels of CD20, there is a
considerable drop-off for CDC mediated by IgG1-7D8 and OFA, but
Hx-7D8 still promotes substantial CDC. Finally, across all of the
CLL B cells examined, the CDC activities of IgG1-7D8 and OFA are
quite similar, in agreement with previous reports (12, 53).
Dose-response tests for CDC in C9-dpl sera differentiate
Hx-7D8 from wild-type complement-fixing mAbs IgG1-7D8
and W6/32
Binding isotherms with the Al555-labeled mAbs (all at very similar
F/P ratios) confirmed that IgG1-7D8 and Hx-7D8 bind to Z138
cells with high avidity, with 50% saturation of binding obtained at
∼1 mg/ml, corresponding to a Kd of 6.7 nM (Fig. 4A). However,
although the avidity of W6/32 (anti-HLA) is somewhat lower
(Kd 46 nM), at saturation ∼6- to –8-fold more W6/32 binds to the
cells compared with the amount of IgG1-7D8 or Hx-7D8 that is
bound, which is consistent with reports that in excess of 300,000
HLA molecules are expressed on the cells (42). The high level of
CDC of Z138 cells mediated by W6/32 at concentrations of
10 mg/ml is likely due to the very high density of mAb W6/32
molecules bound to the cell. Indeed, at this concentration, CDC of
Z138 cells is considerable for all three mAbs in both NHS and in
FIGURE 5. Hx-7D8 has a much higher level of CDC activity than
either IgM-7D8 or IgG1-7D8. mAb dose-response tests with Raji cells
(A), with Z138 cells (B) and with primary CLL cells from one patient (C).
All CDC tests were performed in quadruplicate for 15 min at 37˚C in
25% NHS; comparable results were obtained in 50% NHS (data not
shown). ***p , 0.001.
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On the basis of the high levels of CDC that we observed with
Hx-7D8 and W6/32 in C9-dpl sera, we next explored whether sera
depleted of other single complement components could also
support CDC. There was essentially no mAb-mediated killing of
Z138 cells in sera depleted of complement components up to C5
(Fig. 2B, 2C). However, substantial killing of Z138 cells, mediated
by Hx-7D8 or W6/32, was evident for sera depleted of any one of
the terminal pathway components, and we also found that sera
depleted of complement factor B (fB) or complement factor D
(fD) supported equally high levels of CDC. Similarly, Hx-7D8–
and ALM-mediated CDC of CLL cells in sera depleted of any one
of the terminal pathway components, and CDC was also evident in
sera depleted of fB or fD (Fig. 2D, 2E).
RAPID CDC OF NUCLEATED CELLS
The Journal of Immunology
The enhanced activity of the Hx-7D8 construct is derived most
directly from its increased ability, upon binding to cells, to form
hexamers, bind C1q and thus activate complement (8, 12).
Therefore, we next determined whether IgM-7D8 might also
have a superior level of CDC activity because single molecules of
cell-bound IgM have been demonstrated to have the potential to
activate complement (54). We examined, in dose-response experiments, the CDC activity of the IgM-7D8 against Raji and
Z138 cells as well as against B cells from a CLL patient (Fig. 5).
We find that the CDC activity of IgM-7D8 is relatively modest and
is equal to or less than the activity of the IgG1-7D8. However, in
all cases, as demonstrated in Figs. 1, 3, and 4, Hx-7D8 has significantly enhanced activity compared with IgG1-7D8.
Addition of C9 to C9-dpl sera increases C9 binding but has a
small effect on CDC
FIGURE 6. Reaction of Z138 cells or CLL cells with
Hx-7D8 in C9-dpl sera supplemented with C9 leads to
only modest effects on CDC, but supplementation leads to
a large increase in deposition of the MAC on the cells. (A)
The percentage of CDC for Z138 cells is defined by uptake of TP3. n = 4 in 50% NHS, and n = 8 in 25% NHS.
(B) MAC binding is defined based on probing with FITClabeled mAb aE11; n = 2. (C) As in (A), based on duplicate determinations for CLL cells from four patients
reacted with Hx-7D8. (D) The results for only one CLL
patient are illustrated because there were very large differences between patients in the amount of C9 binding.
However, in three of four CLL patients, p , 0.05 for C9dpl versus NHS. *p , 0.05, **p , 0.01, ***p , 0.001.
A C9 neutralizing mAb blocks CDC of Z138 cells but not CDC
of CLL cells
Our flow cytometry experiments suggest that in C9-dpl sera, at least
part of the CDC observed for mAb-opsonized cells is due to efficient uptake of very small amounts of the MAC, which are
generated from residual C9 in the depleted sera; however, we
cannot yet rule out the possibility that at least some the cells were
killed because of the action of C5b-8. To directly explore this issue,
we determined whether neutralizing Abs specific for C9 could
block CDC either in NHS or in C9-dpl sera. We found that at high
concentrations of the neutralizing anti-C9 mAb 22, in both NHS
and in C9-dpl sera, blockade of C9 inhibits CDC of Z138 cells
induced either by Hx-7D8 or by W6/32 (Fig. 7A, 7C), and this
specific blockade is also accompanied by close to quantitative
reduction in binding of C9 to the cells (Fig. 7B, 7D). Addition of
an isotype control mAb had no effect on CDC or binding of C9.
Competition experiments indicated that the neutralizing anti-C9
mAb 22 did not block binding of FITC anti-C9 mAb aE11 to cells
on which the MAC was already bound under the conditions of
these experiments (data not shown).
When we examined the effect of a similar blockade strategy on
mAb-mediated CDC of CLL cells, we found that CDC was preserved in both NHS and in C9-dpl sera in the presence of high
concentrations of the neutralizing anti-C9 mAb 22 (Fig. 7E, 7G), in
contrast to the blockade observed with Z138 cells. However, in
the presence of the neutralizing anti-C9 mAb, binding of the antiC9 mAb aE11 to the CLL cells was reduced to very close to
background levels in NHS (Fig. 7F). The amount of C9 binding in
C9-dpl sera was 20-fold lower than in NHS in the absence of antiC9 mAb 22 (Fig. 7H) and was further reduced at the highest concentration of anti-C9 mAb 22. On the basis of these findings, we
cannot rule out that the CLL cells may have been killed by C5b-8.
Visualization of cellular intermediates in the CDC pathway
To investigate in more detail the dynamic role of MAC binding in
the CDC process and to identify cellular intermediates generated as
a consequence of MAC binding, we next made use of multispectral
high-resolution fluorescence imaging by flow cytometry (12, 23).
Z138 cells were labeled with the mitochondrial viability dye
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We next determined whether addition of purified C9 to C9-dpl
sera could modulate both CDC and the amount of C9 binding
mediated by the mAbs under investigation. Similar levels of CDC
were observed for Z138 cells reacted with Hx-7D8 in NHS as well
as in C9-dpl sera and in C9-dpl sera supplemented with C9
(Fig. 6A), but addition of C9 to C9-dpl sera led to a large increase in the amount of C9 bound to the cells (Fig. 6B), although
CDC was only marginally increased. This finding is consistent
with our hypothesis that the small amount of C9/MAC that appears to be bound to the cells in C9-dpl sera is adequate to
promote CDC. Addition of a relatively large amount of C9 allows considerably greater numbers of MAC to be generated and
bind to the cells, but they are not absolutely required to promote
cell killing because the killing threshold for MAC binding is
reached even in C9-dpl sera. The modest increase in C9 binding
upon addition of C9 to 25% C9-dpl sera compared with 50% sera
most likely is due to the lower number of C5b-8 complexes
generated.
We conducted similar C9 addition experiments with CLL cells in
C9-dpl sera. Although addition of C9 had virtually no effect on the
amount of CDC mediated by Hx-7D8 in C9-dpl sera (Fig. 6C),
addition of C9 induced considerable increases in the amount of C9
bound to CLL cells reacted with Hx-7D8 (Fig. 6D).
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FIGURE 7. (A–D) Addition of neutralizing anti-C9 mAb 22 reduces CDC of Z138 cells and MAC binding, mediated by Hx-7D8 or W6/32, to background levels in both NHS and in C9-dpl sera. (A and C) The percentage of CDC is defined by uptake of TP3; n = 4. Significant differences are evident for
cells reacted with Hx-7D8, and the isotype control versus cells reacted with Hx-7D8 and the anti-C9 mAb. Comparable differences are evident for cells
reacted with W6/32, but the statistical analyses are omitted for clarity. (B and D) MAC binding is defined based on probing with FITC mAb aE11. Control
experiments with an isotype control in place of mAb 22 showed almost no changes. Background signals were determined on reaction with Hx-b12, in the
presence of anti-C9 mAb 22. Results for only one experiment (n = 2) are shown because there were often substantial differences in absolute MESF values
between experiments. (E–H) Addition of neutralizing anti-C9 mAb 22 has only modest effects on CDC of CLL B cells mediated by Hx-7D8 in both NHS
and in C9-dpl sera (E–G). However, mAb 22 reduces measureable MAC binding to the CLL cells to close to background levels (F–H). The results of
duplicate measurements on CLL cells of a single patient are illustrated, but the same patterns were evident for a second patient (data not shown). *p , 0.05,
**p , 0.01, ***p , 0.001.
The Journal of Immunology
FIGURE 8. Reaction of TMRM-dyed Z138 cells
with CD20 mAbs in 50% NHS leads to rapid CDC,
which is marked first by C3b deposition (A), which
is then rapidly followed by binding of C9 (B–E),
quenching of the TMRM signal (C), and cell death
(D). Data were obtained based on multispectral
high-resolution fluorescence imaging by flow cytometry. The arrows in (A)–(D) at 40 s emphasize that the
deposition of C3b begins prior to binding of C9
or cell death. The arrow is placed in (E) to emphasize that the absolute signal for bound C9 is weak for
cells reacted with Hx-RTX after 60 s, but the results in the other panels demonstrate that the majority of the cells have already reacted and were
killed. In a small-scale replicate study, after 360 s,
mAb 7D8 mediated 98% CDC (TP3 positive), 99%
of the cells were TMRM negative, 98% were C3b
positive, and 92% were C9 positive, in excellent
agreement with the data illustrated in the figure.
Background levels for cells reacted mAb Hx-b12
were also quite low, as expected.
We next applied multispectral high-resolution fluorescence imaging analyses to examine CDC of primary CLL cells mediated by
Hx-7D8 in the presence of NHS, C9-dpl sera, or C9-dpl sera supplemented with C9 (Fig. 10). After reaction, cells were probed with
TP3 and FITC mAb aE11. Because of the very low levels of C9
bound in C9-dpl sera, we did not include the Al405 probe for C3b
deposition to preclude the possibility of a compensation artifact. The
general trends we observed are similar to those illustrated in Figs. 1,
6, and 8. After reaction for 4 min with Hx-7D8 in NHS or in C9-dpl
sera supplemented with C9, the cells are indeed killed and are highly
positive for C9 (Fig. 10B, 10D). However, although cells reacted
with Hx-7D8 for 8 min are also effectively killed in C9-dpl sera, the
C9 signal is quite weak, and we could not identify any individual
cells with a visible green signal (Fig. 10C). This result is consistent
with the observation that the presence of a neutralizing mAb specific
for C9 had little effect on CDC of CLL cells (Fig. 7E, 7G).
To evaluate colocalization of mAb, C3b, and C9, we next reacted
CLL cells with Al546 Hx-7D8 in NHS and then probed with mAbs
specific for C3b and C9. A colocalization score (bright detail
similarity score [BDSS]) for two fluorophores is calculated by
pixel-by-pixel comparison of the two signals on each cell in the
double-positive populations (23, 45). A BDSS . 2.25 indicates the
mAbs are colocalized. In agreement with our previous experiments with CD20 mAbs, we find a very high level of colocalization of C3b with bound mAb (Fig. 11): 77% of the single cells
have a BDSS .2.25 for C3b/mAb colocalization. Colocalization
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TMRM and then reacted for varying times at 37˚C, in NHS, with
several different CD20 mAbs. The cells were washed and probed
with mAbs specific for C3b or C9, and just before analysis, TP3 was
added to measure cell death. The results are presented as percentage
of cells positive for each probe (Fig. 8), and representative images are
displayed in Fig. 9. For each mAb, C3b deposition occurs first and,
soon thereafter, is followed by C9 binding, loss of the TMRM signal
(indicating poisoning of mitochondria by Ca2+), and cell death (uptake of TP3). For example, after 40 s, ∼60% of the Z138 cells reacted
with either Hx-7D8 or Hx-RTX score positive for C3b deposition,
but at this time, only ∼30% of the cells are TMRM negative, ∼30%
are positive for TP3, and ,20% are positive for C9 (see arrows,
Fig. 8A–D). However, even at the 60-s mark, the magnitude of the
FITC C9 signal for cells reacted with any of the mAbs is relatively
modest (arrow, Fig. 8E), although 80% of the cells reacted with
Hx-7D8 or Hx-RTX are killed. However, C9 levels continue to increase after the cells are killed (Fig. 8E, 180 and 300 s).
Cells that are representative of distinct steps in the CDC pathway
are displayed in Fig. 9: unreacted cells at time zero (Fig. 9A); live
cells (TMRM positive and TP3 negative) covalently tagged with
C3b but negative for bound C9 after 40 s of reaction (Fig. 9B); live
cells (TMRM positive and TP3 negative) that have bound C9 after
60 s of reaction (Fig. 9C); dead cells (TMRM negative and TP3
positive) with bound C9 (Fig. 9D); and dead cells C9 dim
(Fig. 9E). Dot plots used to define these populations are shown in
Supplemental Fig. 3.
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RAPID CDC OF NUCLEATED CELLS
of C9 with the mAbs was moderate: 26% of the single cells had a
BDSS for mAb/C9 colocalization .2.25. Finally, we find that
deposited C3b and bound C9 are also partially colocalized: 43%
of the single cells have a BDSS for C3b/C9 colocalization .2.25.
The observation of colocalization of both C3b and C9 with the
mAb and with each other suggests that there is considerable focusing of both complement activation products very close to the
site of mAb binding.
Real-time visualization of binding of C3b and the MAC
To obtain additional insight on the role of C9 in cell killing, we used
four-color confocal microscopy to record real-time movies of the
CDC reaction (Supplemental Videos 1A–D, 2A–D). Z138 cells,
previously dyed with TMRM, were reacted with Hx-7D8 at 25˚C
in NHS supplemented with FITC mAb aE11 and Al405 anti-C3b
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FIGURE 9. Distinct cellular steps in the CDC pathway
are illustrated with representative images from the experiment described in Fig. 6 with Hx-7D8. (A) At zero time,
cells are alive (TMRM positive, TP3 negative). (B) After
40 s, C3b has deposited, but the cells are still alive, based
on the positive TMRM signal and the lack of staining by
TP3. (C and D) After 60 s, C9 has bound to the cells. Note
that both live (C) and dead (D) (TP3 positive, TMRM
negative) C9-positive cells can be seen. (E) After 60 s,
8.6% of dead cells are C9 dim.
3E7. The movies and corresponding screen shots (Fig. 12A) illustrate the following: The earliest event observed was C3b deposition, based on uptake of (blue) Al405 mAb 3E7 at 1–2 min.
Loss of the red TMRM signal is first evident at 2–3 min, but we
were unable to definitively detect any C9 bound to the cells at the
times in which the red TMRM signal was first quenched, likely
because of the high background signal because of the FITC mAb
aE11 in the serum. However, loss of the TMRM signal from cells
was rapidly followed by uptake of TP3 (3–4 min). This is indicative of cell death because of substantial permeabilization of
the cell’s plasma membrane and binding of FITC anti-C9 (green
signal, see arrow, bottom panel, 4 min) is manifest on the cells at a
time approximately coincident with the time at which the purple
TP3 signal is detectable inside the cell. Finally, it is important to
note that even after cells are dead (strong TP3 signal) the intensity
The Journal of Immunology
1771
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FIGURE 10. CLL cells reacted with Hx-7D8 are killed by CDC in NHS as well as in C9-dpl sera and in C9-dpl sera supplemented with C9. Data were
obtained based on multispectral high-resolution fluorescence imaging by flow cytometry. Histograms for FITC C9 binding and representative images
are displayed. (A) Unreacted cells. Three percent of the cells are dead, and 1.3% are C9 positive. (B) After reaction for 4 min with Hx-7D8 in NHS, 92%
of the CLL cells are dead, and 96% are positive for C9. GMI 7.8 3 104. (C) After reaction for 8 min with Hx-7D8 in C9-dpl-sera, 87% of the CLL cells
are dead, and 35% are weakly positive for C9. GMI 1.4 3 103. (D) After reaction for 4 min with Hx-7D8 in C9-dpl sera supplemented with 20 mg/ml
C9, 93% of the CLL cells are dead, and 96% are positive for C9. GMI 12.2 3 104. Similar results were obtained in a separate experiment with CLL B
cells from a different patient. After reaction with mAb Hx-7D8 for 8 min: in NHS, 78% of the cells were dead, and 91% were positive for C9; in C9-dplsera, 54% of the cells were dead, and 6% were weakly positive for C9; in C9-dpl-sera supplemented with C9, 58% of the cells were dead, and 74% were
positive for C9.
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RAPID CDC OF NUCLEATED CELLS
FIGURE 11. C3b and C9 are colocalized on B cells with opsonizing mAb Hx-7D8 after reaction for brief periods in 50% NHS. Data were obtained based
on multispectral high-resolution fluorescence imaging by flow cytometry. CLL cells were reacted with Al546 Hx-7D8 in NHS for 8 min and after two
washes were probed with both FITC mAb aE11 and with Al594 3E7 (for C3b/iC3b). Images representative of triple-positive cells are shown. BDSS values
for the merged images of the individual cells are given. A very high degree of colocalization of Hx-7D8 with C3b is evident, and colocalization of the mAb
with C9 is also observed.
Discussion
The key new finding in this paper is that nucleated cells, including
primary CLL cells, that are reacted with mAbs that fix complement
efficiently, can be killed by CDC at high levels (.80%) and quite
rapidly (,2 min), in the presence of the very small amounts of
residual C9 that are present in C9-dpl sera (Figs. 1, 2, 4, 6, 7 and
10). In particular, Hx-7D8 mediates high levels of CDC of CLL
cells at very low concentrations, in both NHS and C9-dpl sera
(Figs. 4D, 4E, 5).
There are earlier reports indicating that under certain conditions
erythrocytes can be killed by CDC in sera lacking C9 (55). Lint
et al. (56) reported on a C9-deficient individual, and found that
this person’s sera could mediate lysis of sensitized sheep erythrocytes, but the reaction was considerably slower than the rate of
lysis observed in C9-intact normal human serum. This early study
did not address the effect of C9-deficiency on CDC of nucleated
cells. Morgan and coworkers (57) found that a mAb could induce
CDC of U937 cells in C9-dpl sera, but the reaction was slow,
requiring upwards of more than 30 min for lysis.
To our knowledge, the present report constitutes the first detailed
demonstration of individual steps, all the way to MAC binding and
cell death, in the robust and very rapid CDC reaction mediated by
clinically relevant mAbs bound to cell lines and primary CLL cells
in NHS or in sera depleted of C9. In addition, we also find that
during this reaction C9 colocalizes moderately with cell-bound
mAb as well as with covalently deposited C3b (Fig. 11). The
mAbs that mediate these activities include both W6/32 and ALM
as well as the Hexabody molecules specific for CD20. ALM (rat
derived humanized IgG1) and W6/32 (mouse IgG2a, a complement activating subtype) target and bind to epitopes (CD52 and
HLA, respectively) expressed at very high levels on the cell, thus
ensuring a very high level of complement activation due simply to
the high density of mAbs bound to the cell (34, 36, 42). However,
CD20 mAbs Hx-7D8 (but not IgM-7D8, Fig. 5) and Hx-RTX are
able to kill efficiently at lower binding densities due to their enhanced ability to bind C1q upon forming hexamers after binding
to CD20 on the cell (8, 12). These Hexabody molecules also
support rapid and robust C3b deposition and the results presented
here demonstrate that this efficient utilization continues downstream to MAC formation. It is important to note that our results
have been obtained based on analyses that made use of three
different technologies: Flow cytometry, high resolution digital
imaging by flow cytometry, and real-time confocal microscopy
movies. The results using the different techniques are in good
agreement and provide an important validation of our findings. For
example, on the basis of TP3 staining (Figs. 1, 8) and generation
of TMRM-negative cells (Fig. 8), Z138 cells are killed by Hx-7D8
and Hx-RTX in ,2 min. In addition, dead cells with no visible
staining by FITC anti-C9 (C9 dim) could be identified by high
resolution digital imaging (Fig. 9E), indicating that very small
amounts of C9 appear to be adequate to promote cell-killing.
The enhanced ability of Hexabody molecules to activate complement is particularly evident in the dose-response studies. At
lower mAb concentrations (0.33–1 mg/ml), when less mAb is bound
to the cells, the CDC activity of W6/32 as well as IgG1-7D8 are
substantially reduced in both NHS and in C9-dpl sera (Fig. 4).
Under these conditions, Hx-7D8 is still able to promote CDC of
both Z138 cells and CLL cells. The CDC process for Hexabody
mAbs, as well as for the other mAbs at higher concentrations,
would also appear to be quite efficient in making use of limited
resources in C9-dpl sera, that is small amounts of C9. We found
that after cells are killed by CDC in NHS, deposition of C9 appears to continue, although strictly speaking the extra C9 is not
needed, as the cell is already dead (Figs. 8, 9, 12A). This finding is
reinforced in the experiments in which C9 was added back to the
C9-dpl sera. CDC was barely increased, but considerably more C9
was bound to the cells (Figs. 6, 10). This is an important consideration, because it indicates that more complement can be
unnecessarily consumed and exhausted, thus potentially limiting
killing of other mAb-opsonized cells. In other words, one could
say that it is only necessary to kill a cancer cell once, but effector
mechanisms such as complement may continue to turn over, thus
consuming a valuable resource. That is, if large amounts of a mAb
are used to saturate all the binding sites on the cell, then even after
the cell is killed by CDC, complement may continue to be activated by the particulate immune complex, i.e., the dead cell
containing large amounts of bound mAb. Indeed, we detected
continued and increased deposition of C9 on cells after it was
clear that they had already been killed (Fig. 12A).
Thus, our findings have important implications with respect to
the clinical application of these newly developed highly effective
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of the signal because of C9 deposition continues to increase (see
movies and cells at 4–7 min), indicating that the dead cells (which
are essentially particulate immune complexes) continue to activate
complement. Similar patterns were obtained for Z138 cells reacted
with mAb W6/32 (data not shown).
Finally, the results for Z138 cells reacted with Hx-7D8 in C9-dpl
sera (Supplemental Video 2A–D) are notably different from the
results obtained in intact NHS. First, although C3b deposition is
easily demonstrable, many of the cells are not killed, even after an
incubation of 780 s at 37˚C (Fig. 12B). All of the confocal microscopy experiments were conducted at rather high cell concentrations (∼4 3 107 cells/ml), and as noted earlier, CDC in
C9-dpl sera is reduced at this high cell burden. In addition, where
cell killing is observed, the kinetics of Hx-7D8–mediated CDC at
37˚C in C9-dpl sera are clearly slower than the rate of killing
observed for cells reacted with Hx-7D8 at 25˚C in NHS. Moreover, we cannot detect any evidence of C9 binding on the cells that
are killed in C9-dpl sera.
The Journal of Immunology
1773
complement-activating Hx-mAbs. We have previously reported
that at the usual high doses of RTX or OFA used in the treatment of
CLL, complement and other effector mechanism can be exhausted,
thus leading to a perfect storm in which mAb-opsonized cells are
not cleared by effector functions, but instead CD20 is removed
from the cells due to trogocytosis (24, 58), thereby further
compromising mAb immunotherapeutic efficacy. We now demonstrate that at quite low concentrations the Hexabody mAbs can
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FIGURE 12. Four-color confocal fluorescence
microscopy analyses of the kinetics of CDC of
Z138 cells with an emphasis on MAC binding: (A)
CDC mediated by Hx-7D8 at room temperature in
NHS. Images are derived from Supplemental
Video 1A–D. Images displayed are based on
analyses with two colors at advancing times during the reaction. Upper panel, Al405 mAb 3E7
(C3b/iC3b, light blue) and TMRM (viable mitochondria, red). The arrows at 2 and 3 min indicate
C3b deposition, followed by TMRM quenching,
respectively. Middle panel, Al405 mAb 3E7
(C3b/iC3b) and TP3 (dead cells, bright purple).
Lower panel, FITC anti-C9 (green) and TMRM
(viable mitochondria, red). Bottom panel, FITC
anti-C9 (green) and TP3 (dead cells, bright purple). The arrow at 4 min denotes the first appearance of FITC mAb aE11 binding. (B) CDC
mediated by Hx-7D8 at 37˚C in C9-dpl sera. The
images are derived from Supplemental Video
2A–D. Fluophores as in (A). The arrow in the
upper panel at 78 s denotes C3b deposition. The
arrows at 468 s in the upper and next panel denote
TMRM quenching and cell killing, respectively.
Representative of at least three movies each for
Supplemental Videos 1 and 2.
promote robust CDC of CLL cells even in sera depleted of late
complement components, and the mAbs also promote substantial
CDC of CLL cells with low CD20 levels (Fig. 3). On this basis, it
should be quite feasible to initiate mAb treatment regimens
based on relatively low mAb doses (∼30–40 mg) ∼3 times per
week (59–62). Circulating cells should be efficiently eliminated
by several mechanisms, especially as a consequence of C3b
opsonization and CDC, and complement activities will be better
1774
Acknowledgments
We thank the following individuals for invaluable advice and assistance:
Dr. Yalin Wang and Dr. Stacey Guillot of the University of Virginia
(UVA) Advanced Microscopy Facility, Joanne Lannigan of the UVA Flow
Cytometry Core Facility, Dr. Mark Conaway of the UVA Public Health
Sciences Division, and Karl VanDerMeid at the University of Rochester.
We thank the CLL patients for kindly donating blood samples for this
research, and we thank the Wilmot Foundation for support of the tumor
bank at the University of Rochester.
Disclosures
H.v.d.H, S.O., F.J.B., J.S., and P.W.H.I.P. are employees of Genmab. R.P.T.
received funding from Genmab. The other authors have no financial conflicts of interest.
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preserved for repeated infusions a few days later. Moreover, therapies based on such low-dose treatments could be accomplished
based on s.c. infusions, instead of i.v. infusions, which would be far
more convenient for both patients and health-care providers.
On the basis of an ELISA, we estimate that the concentration of
residual C9 in C9-dpl sera is #250 ng/ml. Because the concentration of C9 in intact NHS is 60 mg/ml (48), this indicates that
.99% of the C9 was indeed removed from the NHS. However,
calculation of the minimum C9 concentration needed to kill nucleated cells is most instructive: In our flow cytometry experiments, the usual cell concentration was 5 3 106 cells/ml. If we
assume that at least 10,000 MAC are needed to bind to a nucleated
cell to kill it (13, 14) and that there are 10 C9 molecules per MAC
(63), then for this cell burden a C9 concentration of 5 3 1011
molecules/ml in 25% C9-dpl sera, corresponding to 60 ng/ml
C9 (m.w. 70 kDa, 240 ng/ml in neat serum) would be sufficient
to kill the cell. Thus, there should be enough C9 in the C9-dpl sera
to mediate CDC. This calculation presumes that the affinity of
C9 for the cell-associated C5b-8 complex is very high and that the
small amount of C9 in the serum can therefore be quantitatively
bound to C5b-8 on the cells. The key role of residual C9 in promoting CDC of the Z138 cells is emphasized by the action of C9
neutralizing mAb 22, which blocked CDC in both NHS and in
C9-dpl sera (Fig. 5).
Additional evidence for the efficacy of the mAbs with respect to
use of complement derives from the colocalization studies
(Fig. 11). Indeed a considerable amount of C9 bound to the cell is
found in close proximity to bound mAb and C3b, suggesting that
it can be tightly focused on the cell, thus generating cytotoxic
pores, even when relatively small amounts of C9 are bound. The
mAbs are also able to promote CDC in sera depleted of C6, C7, or
C8 (Fig. 2) and we suggest that similar findings will be obtained
when other next-generation mAbs are examined under conditions
making use of limiting amounts of these complement components.
In addition, although CDC was abrogated in C1q-dpl sera, CDC
mediated by Hx-7D8 and ALM was quite high in sera lacking fB
or fD, suggesting that for some mAbs the classical pathway of
complement is key for mAb-mediated CDC and that amplification
by the alternative pathway may not make a significant contribution
to CDC.
The question as to whether CLL cells are susceptible to CDC in
the complete absence of C9 remains uncertain. We made numerous
efforts to obtain sera from individuals genetically deficient in C9,
but we were not successful. However, our results do demonstrate
that CLL cells can indeed be killed by CDC in the presence of very
low levels of active C9.
In summary, we report that several different mAbs can activate
complement and promote CDC, on cell lines and on primary
CLL cells, in sera immunologically depleted of late complement
components. We have focused our efforts on C9-dpl sera, and our
results suggest that the trace, residual amount of C9 remaining in
the sera is responsible for much of the CDC activity. CD20
mAb Hx-7D8 is particularly effective at activating complement
after it binds to a cell and its activity is even demonstrable at
relatively low concentrations in C9-dpl sera when only a fraction
of the CD20 sites are chelated by the mAb. This activity is also
manifest in colocalization experiments, which indicate that cellbound Hx-7D8 can focus complement components C3b and C9
very close to its binding sites on cells, thus making particularly effective use of complement and allowing for CDC in sera
containing just trace amounts of C9. Strategies that target other
sites on malignant cells that also take advantage of the hexamerformation paradigm may prove to be quite effective in the immunotherapy of cancer.
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