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NEOPLASIA
From the Cancer Sciences Division, School of Medicine,
General Hospital, Southampton, United Kingdom; Department of Medicine
III, University of Erlangen-Nurnberg, Nuremberg, Germany; and
Department of Immunology and Medarex Europe, University Medical Center,
Utrecht, The Netherlands.
CD64 (Fc Recent clinical trials have generated considerable
optimism for the use of monoclonal antibodies (mAb) in a range of
neoplastic diseases.1 Two reagents in particular are
behind the current enthusiasm, chimeric anti-CD20 (Rituximab), used in
relapsed non-Hodgkin lymphoma,2 and humanized anti-Her-2
(Herceptin), which reacts with the proto-oncogene erbB2 and is used in
metastatic breast cancer.3 Both products are relatively
nontoxic and provide benefits to patients. Perhaps the most interesting
aspect of this work arises from recent evidence showing that when mAb
are used in combination with certain cytotoxic drugs or radiotherapy,
the therapeutic efficacy, but not the toxicity, of the 2 moieties are
additive or even synergistic.4
Despite such successes we are still surprisingly ignorant of how mAb
operate in vivo.2,4-8 Direct transmembrane signaling to
the tumor cell, recruitment of cellular effectors, and activation of
complement-dependent lysis have all been implicated as possible mechanisms. Recent in vivo evidence underlines the importance of FcR in
mediating mAb function and shows that not only are FcR essential for
mAb anticancer activity, but that they can act as inhibitory molecules
on certain effector cells and significantly diminish killing
activity.9,10 Thus, under normal circumstances, the
therapeutic activity of any particular mAb will be determined by a fine
balance of inhibitory and stimulatory signals, controlled by different
FcR and dependent on the mAb and its isotype.
In the current study we have investigated the potential of a panel of
BsAb to retarget polymorphonuclear cells (PMN) against lymphoma through
their Fc Because there are no mAb available for mouse CD64, until now it has not
been possible to test CD64 BsAb in syngeneic tumor models. The
generation of CD64 transgenic (Tg) mice, expressing the human FcR as a
transgene under the control of the endogenous regulatory elements, has
overcome this limitation.16,17 Here we show for the first
time direct evidence that CD64-directed BsAb are highly therapeutic in
vivo, are more active than IgG mAb, and that certain BsAb can provide
long-term protection to tumor-bearing animals.
Animals and cell lines
All cell cultures were grown in supplemented Dulbecco modified Eagle
medium (DMEM) (Life Technologies, Paisley, UK) containing glutamine (2 mmol/L), pyruvate (1 mmol/L), penicillin and streptomycin (100 IU/mL), Fungizone (2 µg/mL), and 5% to 10% fetal calf serum (FCS; Myoclone; Life Technologies), except Monoclonal and bispecific antibodies
F(ab')2 fragments were prepared from IgG by limited
proteolysis with pepsin at pH 4.1 to 4.2 in 0.1 mol/L sodium
acetate.26 Bispecific F(ab')2 heterodimers
were prepared using o-phenylenedimaleimide (o-PDM; Sigma, UK).26,27 The
constructs were size fractionated and passed through an anti-Fc Flow cytometry to determine huCD64 expression Whole blood obtained from tail bleeds of 6-week-old mice was analyzed by direct immunofluorescence staining using a FACSCalibur (Becton Dickinson, San Jose, CA). Samples were incubated with fluorescein isothiocyanate (FITC)-labeled M22 (50 µg/mL) for 20 minutes on ice, treated once with FACS-lysing solution (Becton Dickinson) for 1 minute, then washed with phosphate-buffered saline (PBS) containing 1% bovine serum albumin, and analyzed for expression of huCD64 by gating on the PMN population as determined by FSC/SSC profiles.Redirected cellular cytotoxicity assay In vitro cytotoxicity was measured using a standard chromium 51Cr-release assay.7 Briefly, 51Cr-labeled target cells (A31 or BCL1) were prepared from spleens of terminal animals and resuspended at 105/mL in supplemented RPMI. The effectors were murine PMN from whole blood separated on a discontinuous Percoll (Sigma) gradient.28 To mobilize PMN, mice received 2 µg/d recombinant murine G-CSF (Amgen, Thousand Oaks, CA) subcutaneously for 4 days before PMN isolation. After separation PMN were washed once and resuspended in supplemented RPMI. For the assay, 50 µL aliquots of target cells (5 × 103) and BsAb were mixed in wells of a 96-well U-bottomed plate (Life Technologies) and left on ice for 15 minutes. Aliquots (100 µL) of effector cells were then added at an effector:target (E:T) ratio of 50:1. Plates were centrifuged at 200g for 5 minutes at room temperature, incubated for 4 hours at 37°C, and recentrifuged at 500g for 5 minutes before 100 µL of supernatant was harvested from each well to estimate the released 51Cr.Determinations were in triplicate, and maximal release of
51Cr was obtained using target cells to which 150 µL of
1% Nonidet P-40 had been added. The percentage specific
51Cr release was calculated using the formula:
[(sample release Immunotherapy Groups of age-matched F1 mice were injected intraperitoneally with 105 BCL1 (BALB/c × FVB/n) or 2 × 105 A31 (CBA × FVB/n) lymphoma cells. If appropriate, mice also received 2 µg/d murine G-CSF subcutaneously to mobilize PMN, administered daily for 10 days from 4 days before to 5 days after the tumor inoculum. For most experiments mice were treated with BsAb (intraperitoneally) twice daily on days 1 to 5 after the tumor. Both tumors develop primarily in the spleen, with a leukemic overspill toward the end of the disease.18,19 Lymphoma-bearing animals surviving for more than 100 days after treatment with BsAb were rechallenged, along with age matched naive mice, with 104 fresh tumor cells. Survival was monitored daily, and the results were analyzed using the 2 test of
Peto.29 Animal immunotherapy was cleared through the local
ethical committee and was performed under Home Office license.
Tracking tumor cells in vivo To investigate the activity of BsAb early in the course of tumor development, groups of mice were inoculated with 107 BCL1 tumor cells intraperitoneally on day 0 and then treated with 50 µg BsAb intraperitoneally twice daily, on days 1 to 5 (500 µg/mouse in total). If necessary mice also received murine G-CSF (2 µg/d) as before. Mice were killed on consecutive days throughout the course of therapy, and peritoneal cells were removed by lavage with PBS. Cells were washed once by centrifugation and resuspended in supplemented RPMI at 106/mL.Tumor cells in the exudate were detected by dual-fluorescence staining using FITC-NIMR6 (rat anti-mouse CD22)30 and phycoerythrin (PE)-Mc10-6A5 (rat anti-BCL1 Id). Cells were stained with mAb (50 µg/mL) on ice for 15 minutes. In the case of mice treated with anti-Id mAb, which caused blocking or modulation of the surface Id, cells were stained with FITC-NIMR6 (rat anti-mouse CD22) and PE-ID3 (rat anti-mouse CD19) instead. After staining, cells were treated with FACS lysis solution to lyse red blood cells and then washed with PBS containing 1% bovine serum albumin To detect the presence of tumor cells in the spleen, homogenates were washed by centrifugation, and cells were resuspended in supplemented RPMI at a concentration of 106/mL. Cells then were stained using either FITC-NIMR6 versus PE-Mc10-6A5 to detect BCL1 cells or FITC-M22 (anti-human CD64) and PE-7/4 (rat anti-mouse neutrophil) (VH Bio, Newcastle Upon Tyne, UK) to detect PMN. Measurement of intracellular free calcium Intracellular free calcium concentration was measured using the cell-permeable fluorescent probe Indo-1-AM (Sigma). Indo-1-AM gives a shift in fluorescence emission from 482 nm to 398 nm on calcium binding, and an increase in the ratio of the 398 nm/482 nm signal is indicative of increased intracellular free calcium. Cultured BCL1 cells (107/mL)
were washed twice in serum-free media and loaded with
Indo-1-AM at 5 µmol/L for 30 minutes at 37°C. Cells
were then washed twice by centrifugation in serum-free RPMI to remove
excess dye and were resuspended in supplemented RPMI at
5 × 106 cell/mL. Labeled cells were stored in the dark
at room temperature until used. For the assay, 250 µL of this cell
suspension was warmed to 37°C for 5 minutes, an equal volume of mAb
solution added at a final concentration of 2 µg/mL, and immediately
analyzed using flow cytometry on a FACS Vantage (Becton Dickinson).
Where appropriate, hyper-cross-linking sheep anti-rat IgG or rabbit antihamster IgG was added at a final concentration of 20 µg/mL immediately before analysis. Intracellular calcium mobilization was
estimated by determining the mean fluorescence ratio of Indo-1 bound
(398 nm, FL5) to free calcium (482 nm, FL4) against time. The response
was measured over 300 seconds and recorded using the Cell Quest program
(Becton Dickinson).
Expression of human Fc  RI/CD64 were generated
with its transgene under the control of endogenous regulatory elements.16 This results in normal CD64 expression
patterns on monocytes/macrophages, dendritic cells, and PMN, and
responsiveness to stimulation with G-CSF. F1 mice (FVB/n × BALB/c
and FVB/n × CBA), compatible for the growth of BCL1
(BALB/c) and A31 (CBA) lymphoma, were screened for the expression of
huCD64 on circulating PMN by flow cytometry. The 50% of animals
expressing the transgene were selected for immunotherapy, and non-Tg
littermates were controls. To confirm that huCD64 expression on PMN was
responsive to G-CSF in the F1 mice, we examined levels before and after
4 days of treatment with cytokine. Figure
1 shows expression in a non-Tg mouse (A),
an untreated huCD64 Tg mouse (B), and a G-CSF-primed Tg mouse (C).
Treating mice with G-CSF increased in the level of huCD64 expression on
PMN. Although the expression of huCD64 could be increased with low
levels of G-CSF (eg, 0.5 µg/d), the total number of circulating PMN
was increased (from approximately 14% to 50%) only when mice were
given 2 µg/d (Figure 1; inset). For this reason the treatment
protocol included 2 µg G-CSF/d for all subsequent experiments.
Redirected cellular cytotoxicity of A31 and zcaseBCL1 Considerable evidence suggests that, at least in human systems, CD64 is a potent trigger for PMN.31 To determine the cytotoxic potency of in vivo-activated mouse PMN we performed redirected cellular cytotoxicity (RCC) assays using huCD64-specific BsAb directed at 3 tumor targets, [huCD64 × Id], [huCD64 × CD19], and [huCD64 × MHC II]. Figure 2 shows that activated PMN were able to lyse both A31 and BCL1 tumor cells in the presence of all 3 BsAb. However, MHC II-directed PMN clearly gave the highest levels of lysis, in particular against A31, when RCC resulted in more than 90% specific 51Cr-release. Tumor cells were not destroyed in the presence of PMN from non-Tg animals (huCD64-negative littermates) that were either unprimed or given G-CSF. This indicates that BsAb were indeed functioning by binding huCD64 on PMN. Importantly, tumor cells were also not lysed by PMN taken from huCD64 Tg mice that had not received G-CSF, presumably because these PMN were not activated and FcRI levels were relatively low.
Immunotherapy of A31- and BCL1-bearing mice Having established that our BsAb could mediate tumor killing in the presence of G-CSF-primed PMN, especially when targeted through MHC II, we next determined their efficacy in vivo. The therapeutic model was based on previous work from this32 and other33 laboratories, giving twice-daily treatments with BsAb to compensate for the short half-life of F(ab')2 derivatives. Figure 3 illustrates an initial set of immunotherapy experiments obtained when lymphoma-bearing (A31 or BCL1) mice were treated with G-CSF and anti-Id-based derivatives. Interestingly, the [huCD64 × Id] BsAb provided complete tumor protection for almost all mice, even when used at a dose of just 50 µg/mouse. This result shows that CD64 is an effective trigger molecule for BsAb-based therapy in vivo. By contrast, neither anti-Id F(ab')2 nor a mixture of anti-Id and anti-huCD64 F(ab')2 provided any protection, demonstrating that only when the 2 Ab specificities are linked to form a bispecific unit does effector retargeting occur effectively.
We next asked whether the nontumor-specific derivatives
([huCD64 × CD19] or [huCD64 × MHC II] BsAb) were also active
in vivo. Figure 4 shows that these
derivatives conferred a small survival advantage over control cohorts
that was statistically significant in the BCL1 model
(P < .02) but not in A31. However, the degree of
protection observed was considerably less than that given by [huCD64 × Id] BsAb and, consequently, did not correlate with the derivatives' in vitro activity. In particular, the [huCD64 × MHC II] BsAb, which was exceptionally potent in vitro, showed surprisingly little activity in these therapeutic models. This relative lack of in
vivo efficacy did not result from giving insufficient [huCD64 × MHC
II] BsAb because protection was not improved even when the dose was
increased to 1 mg/mouse (not shown). The possibility that
[huCD64 × MHC II] BsAb was eradicating an important population of
MHC II-expressing effector cells was also investigated using a mixture
of [huCD64 × MHC II] and [huCD64 × Id] BsAb. In these experiments the Id-directed BsAb still displayed full therapeutic activity, suggesting that this was not occurring (not shown).
As expected no therapeutic activity was observed with any of the BsAb
in non-Tg littermates (with or without G-CSF) or in Tg mice that had
not received cytokine treatment. Figure 5
shows an example of these controls for the BCL1 model, with
the data from Tg and non-Tg mice treated with or without G-CSF and
either IgG or BsAb anti-Id. At the antibody level used (50 µg/mouse
total) the IgG anti-Id was not therapeutic in any of the mice, but the BsAb protected huCD64 Tg mice given G-CSF. These results confirm the
importance of huCD64 expression to BsAb activity and, interestingly, show for the first time that the BsAb is more active in vivo than IgG
mAb.
To investigate this latter observation further, we compared the
efficacy of BsAb and IgG anti-Id mAb in the BCL1 model
using BsAb and IgG anti-Id at a range of doses. Figure
6 shows the results for cohorts of
huCD64-Tg mice given G-CSF, 105 BCL1, and then
treated with 1 µg to 1000 µg anti-Id IgG or [huCD64 × Id]
BsAb. These results underscore the superior activity of BsAb in mice.
This is most clearly seen with 1000 µg (Figure 6D) where,
though the anti-Id IgG provided approximately 25 days of protection, treatment with the BsAb resulted in long-term survival for
all animals. Similar observations were found in the A31 model (data not
shown).
Tumor tracking in vivo Because there were discrepancies between the cytotoxic activity of BsAb in vitro compared with their in vivo therapeutic effect, we next investigated their in vivo activity more closely. Tumor tracking experiments were set up to measure the early and late activity of each derivative in the peritoneal cavity and spleen. To monitor the tumor cells, it was necessary to inoculate mice with 107 BCL1 cells/animal, and consequently the dose of BsAb was increased (500 µg/mouse from days 1-5). Early therapeutic activity in the peritoneal cavity was measured by detecting tumor cells in PBS lavage using flow cytometry. Figure 7 represents a typical set of observations. Interestingly, in the first 24- to 48-hour period after treatment, all 3 BsAb ([huCD64 × Id], [huCD64 × CD19], and [huCD64 × MHC II]) were able to dramatically deplete the peritoneal tumor load to less than 1% of that in controls. The CD19-specific BsAb appeared the least active, requiring 48 hours to deplete the peritoneal cavity. Tumor cell depletion was not observed in non-Tg littermates (data not shown), again indicating the requirement for huCD64. Both the anti-Id and anti-MHC II derivatives were particularly potent, showing maximal reduced tumor volume by 24 hours.
Despite this dramatic reduction in the number of tumor cells
recovered during the early course of tumor development, with one
exception, all groups of mice had developed a large splenic tumor load
by day 15 (Figure 8). The exception was
the group treated with [huCD64 × Id] BsAb. By day 15 these animals
contained relatively few splenic tumor cells; furthermore, in parallel
experiments, it was found that they survived significantly longer than
controls despite having received an inoculum of 107 tumor
cells (not shown).
Calcium signaling after hyper-cross-linking of BsAb Several reports indicate that cross-linking the B-cell antigen receptor (BCR) with anti-Ig on various types of B cell generates antiproliferative signals leading to cell cycle arrest (CCA) or apoptosis and that these require the release of intracellular calcium.6,34 To determine whether [huCD64 × Id] BsAb was capable of causing similar responses in mouse tumor cells, we next measured intracellular calcium fluxes after short-term treatment in culture. The variant cell line,BCL1, was labeled with Indo-1-Am and then exposed to
BsAb at a final concentration of 2 µg/mL before measuring calcium
mobilization by flow cytometry. Figure 9
shows calcium fluxes in cells cultured with the various BsAb
derivatives either alone or when cross-linked by polyclonal anti-Ig
serum. As expected, without cross-linking none of these univalent
derivatives was capable of generating a calcium flux. However, whereas
in the presence of a polyclonal anti-Ig serum the [huCD64 × Id]
BsAb-treated cells showed a sharp mobilization of intracellular
calcium, the [huCD64 × MHC II] and [huCD64 × CD19]
derivatives were still without effect.
Lymphoma treatment with [CD64 × Id] BsAb leads to T-cell immunity A surprising feature of the current work with [huCD64 × Id] BsAb was the lack of Id-negative variants emerging in long-term survivor mice. Such escape variants have been a common feature and a disadvantage of anti-Id mAb therapy since it was first investigated in the early 1980s.35,36 To examine whether this is related to the establishment of long-term immunity, we next rechallenged mice that had survived 100 days or more after tumor and [huCD64 × Id] with 104 fresh lymphoma cells (intraperitoneally). To date, 7 mice have been rechallenged (2 with BCL1 and 5 with A31), and, interestingly, unlike naive mice that developed lymphoma as normal, all the "BsAb-cured" mice were completely resistant to this rechallenge and remained disease free for another 100 days.To determine the importance of T cells in the early phases of the
BsAb therapy, we next tested [huCD64 × Id] therapy in mice depleted of CD4 or CD8 T cells, or both. The results clearly show (Figure 10) that though 70% of
T-cell-intact mice were protected from BCL1 by this BsAb,
depletion of the CD4 or CD8, or both, removed almost all its
therapeutic efficacy. It is important to note that in this system,
BCL1 lymphoma develops with the same kinetics in
T-cell-depleted mice as in intact mice.8 Thus we can
conclude that [huCD64 × Id] therapy is dependent on both CD4 and
CD8 cells for its long-term therapeutic activity. This is an important
observation because it points to a likely explanation for the potency
of [huCD64 × Id] compared to the other BsAb and the anti-Id IgG,
which are unable to initiate effective T-cell responses.
In this work we have shown for the first time that huCD64-directed
F(ab')2 BsAb can efficiently eradicate lymphoma cells and generate T-cell immunity in tumor-bearing mice and that such reagents are more potent than IgG mAb. Furthermore, with one reagent,
[huCD64 × Id] BsAb, long-term survival was reproducibly achieved
in 2 B-cell lymphomas (BCL1 and A31) In view of the clinical success achieved with IgG anti-Id mAb in patients with non-Hodgkin lymphoma38 and the potency of [CD3 × Id] BsAb in controlling B-cell lymphoma in mouse models,39 the therapeutic activity of the [huCD64 × Id] BsAb derivatives reported here is perhaps not surprising. Accumulating evidence suggests that much of the success of IgG anti-Id mAb relates to their ability to generate antiproliferation signals.5,6 Extensive literature shows that engaging the BCR with anti-Id, anti-IgM, or antigen itself can trigger a cascade of biochemical changes that control the growth of normal and neoplastic B cells.4,40,41 Given its relative in vitro and in vivo potency and the unique ability of [huCD64 × Id] BsAb to mobilize intracellular calcium, we propose that this reagent probably operates by a number of mechanisms, including RCC, common to all 3 BsAb; direct growth control through transmembrane signaling of the BCR; and the induction of T-cell immunity (below). The relative contribution of these processes remains to be determined. However, a BsAb, because it is univalent for its target molecules on the effectors and tumor cells, can only generate a transmembrane signal(s) when it is cross-linked by multivalent cell surfaces. Thus, to achieve the signaling component of their therapeutic activity, BsAb directed at the Id require effector cells, not only for their cytotoxic capacity but also as multivalent surfaces to provide hyper-cross-linking. Interestingly, the fact that F(ab')2 fragments from anti-Id mAb are inactive in vivo (Figure 3) indicates that the degree to which neighboring BCR molecules are cross-linked at the cell surface is critical for triggering such growth control and suggests that both BsAb and IgG anti-Id derivatives require effector-cell cross-linking to achieve therapeutic activity.42 The current data show that in these models at least, BsAb ([huCD64 × Id]), in combination with G-CSF, is more therapeutically active than anti-Id IgG. The explanation for such potency seems to lie in the ability of this BsAb to generate a potent T-cell response. Mice that had achieved long-term survival after BsAb treatment were resistant to tumor rechallenge 100 days later, indicating that the immunity was robust and long lasting. Furthermore, depletion of either the CD4 or the CD8 cells before and during BsAb therapy removed almost all the efficacy of the BsAb. This is a very exciting development that has not been observed with other anti-Id-based therapies. Why the [huCD64 × Id] BsAb has this unique activity is unclear and is the subject of intensive ongoing investigations. This finding has brought forth a wealth of new questions relating to the mechanisms leading to immunity and the identity of the tumor antigens being recognized. Although CD64 has been shown to be highly active in targeting antigens for immunization,43 in the current work it does not automatically lead to tumor immunity because the 2 other CD64-specific BsAb did not show such activity. Therefore, we can conclude that immunization is dependent on the anti-Id specificity (Figure 9). One possibility is that [huCD64 × Id] BsAb signals through the BCR and induces a form of cell death in the lymphoma that is conducive to a T-cell response. For example, because anti-Id based reagents are likely to induce extensive apoptosis of lymphoma cells, these are probably engulfed and then cross-primed by antigen-presenting cells, such as activated dendritic cells, leading to the subsequent antitumor CTL response.44,45 In contrast, perhaps the other BsAb are less apt to induce apoptosis of target cells in vivo but promote direct phagocytosis by CD64 with or without myeloid cells. Finally, we should not overlook the possibility that cross-linking the BCR with BsAb, by triggering in a manner somewhat akin to multivalent antigen, probably results in a surge of B-cell lymphoma-derived cytokines and other immunologic factors, all of which will create a general inflammatory milieu conducive to a T-cell response. The fact that [huCD64 × Id] cleared most of the lymphoma cells from the peritoneal cavity in 24 hours (Figure 7) suggests that if the lymphoma does release inflammatory mediators, the opportunity for this to occur is very short. In addition to explaining why [huCD64 × Id] is more potent
than the other BsAb, we must also consider its activity relative to
that of the anti-Id IgG. The anti-Id IgG consistently gives a modest
protection of lymphoma-bearing mice, but it was unable to provide
long-term T-cell, protection. There are a number of reasons why the
anti-Id IgG would not be as active as the BsAb in this respect. First,
both anti-Id IgG reagents (A31 and BCL1) were of the rat
IgG2a isotype, which is known not to be particularly effective at
binding to mouse FcR. Second, because the BsAb binds to an epitope on
the Fc For the future, huCD64-directed BsAb in conjunction with the
appropriate activating cytokine appears a highly flexible and effective
treatment option for lymphoma and probably other neoplastic diseases.
The current work underscores the importance of target selection and
suggests that molecules that trigger appropriate transmembrane signals
in the target may be more effective than those simply able to recruit
cytotoxic effectors. Whatever mechanisms BsAb use in vivo, including
recruitment of cytotoxic effectors, cross-linking of signaling
molecules, and potentiation of host antitumor responses, it appears
they are considerably more active than unmodified IgG mAb. Other
candidate molecules that might provide interesting targets on NHL are
CD79
We thank all the members of the Tenovus Cancer Laboratory, who provided expert technical support. We thank Dr Mark Cragg, Dr Ruth French, Dr Heidi van Ojik, and Dr Aymen Al-Shamkhani for valuable input and discussion. We also thank J. Andresen (Amgen) for generously providing recombinant murine G-CSF.
Submitted December 10, 1999; accepted July 19, 2000.
Supported by the European Union, Tenovus of Cardiff, the Cancer Research Campaign, and the Leukaemia Research Fund. T.V. is funded by Deutsche Forschungsgemeinschaft (Va 124/1-3).
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Martin J. Glennie, Tenovus Laboratory, Cancer Sciences Division, School of Medicine, General Hospital, Tremona Rd, Southampton, SO16 6YD United Kingdom; e-mail: mjg{at}soton.ac.uk.
1. Weiner LM. An overview of monoclonal antibody therapy of cancer. Semin Oncol. 1999;26:41-50[Medline] [Order article via Infotrieve].
2.
Maloney DG, Grillo-Lopez AJ, White CA, et al.
IDEC-C2B8 (Rituximab) anti-CD20 monoclonal antibody therapy in patients with relapsed low-grade non-Hodgkin's lymphoma.
Blood.
1997;90:2188-2195
3.
Cobleigh MA, Vogel CL, Tripathy D, et al.
Multinational study of the efficacy and safety of humanized anti-HER2 monoclonal antibody in women who have HER2-overexpressing metastatic breast cancer that has progressed after chemotherapy for metastatic disease.
J Clin Oncol.
1999;17:2639-2648 4. Cragg MS, French RR, Glennie MJ. Signaling antibodies in cancer therapy. Curr Opin Immunol. 1999;11:541-547[Medline] [Order article via Infotrieve].
5.
Vuist WMJ, Levy R, Maloney DG.
Lymphoma regression induced by monoclonal anti-idiotypic antibodies correlates with their ability to induce Ig signal transduction and is not prevented by tumor expression of high levels of Bcl-2 protein.
Blood.
1994;83:899-906
6.
Vitetta ES, Uhr JW.
Monoclonal antibodies as agonists: an expanded role for their use in cancer therapy.
Cancer Res.
1994;54:5301-5309
7.
Tutt AL, French RR, Illidge TM, et al.
Monoclonal antibody therapy of B cell lymphoma: signaling activity on tumor cells appears more important than recruitment of effectors.
J Immunol.
1998;161:3176-3185 8. French RR, Chan HTC, Tutt AL, Glennie MJ. CD40 antibody evokes a cytotoxic T-cell response that eradicates lymphoma and bypasses T-cell help. Nat Med. 1999;5:548-553[Medline] [Order article via Infotrieve]. 9. Ravetch JV. Fc receptors. Curr Opin Immunol. 1997;9:121-125[Medline] [Order article via Infotrieve]. 10. Clynes RA, Towers TL, Presta LG, Ravetch JV. Inhibitory Fc receptors modulate in vivo cytoxicity against tumor targets. Nat Med. 2000;6:443-446[Medline] [Order article via Infotrieve]. 11. Segal DM, Weiner GJ, Weiner LM. Bispecific antibodies in cancer therapy. Curr Opin Immunol. 1999;11:558-562[Medline] [Order article via Infotrieve].
12.
Deo YM, Graziano RF, Repp R, van de Winkel JGJ.
Clinical significance of IgG receptors and Fc
13.
Stockmeyer B, Valerius T, Repp R, et al.
Preclinical studies with Fc
14.
van Ojik HH, Gronewegen G, Repp R, Valerius T, van de Winkel JGJ.
Clinical evaluation of the bispecific antibody MDX-H210 (anti-HER-2/neu × anti-Fc 15. Pullarkat V, Deo Y, Link J, et al. A phase I study of a HER2/neu bispecific antibody with granulocyte-colony-stimulating factor in patients with metastatic breast cancer that overexpresses the HER2/neu. Cancer Immunol Immunother. 1999;48:9-21[Medline] [Order article via Infotrieve].
16.
Heijnen IAFM, van Vugt MJ, Fanger NA, et al.
Antigen targeting to myeloid-specific human Fc 17. Heijnen IAFM, Rijks LJM, Schiel A, et al. Generation of HER-2/neu-specific cytotoxic neutrophils (PMN) in vivo. J Immunol. 1997;159:5629-5639[Abstract]. 18. Slavin S, Strober S. Spontaneous murine B cell leukemia. Nature. 1998;272:624-626. 19. Cobb LM, Glennie MJ, McBride HM, Breckon G, Richardson TC. Characterisation of a new B cell lymphoma. Br J Cancer. 1996;54:807-818.
20.
Tutt AL, Reid R, Wilkins BS, Glennie MJ.
Activation and preferential expansion of rat cytotoxic (CD8) T cells in vitro and in vivo with a bispecific (anti-TCR
21.
Illidge TM, Cragg MS, McBride HM, French RR, Glennie MJ.
Radioimmunotherapy of B cell lymphoma: the importance of antibody specificity in determining successful treatment of established disease.
Blood.
1999;94:233-243 22. George AJT, McBride HM, Glennie MJ, Smith LJ, Stevenson FK. Monoclonal antibodies raised against the idiotype of the murine B cell lymphoma, BCL1, act primarily with heavy chain determinants. Hybridoma. 1991;10:219-227[Medline] [Order article via Infotrieve]. 23. Krop I, de Fougerolles AR, Hardy RR, Allison M, Schlissel MS, Fearon DT. Self renewal of B-1 lymphocytes is dependent on CD19. Eur J Immunol. 1996;26:238-242[Medline] [Order article via Infotrieve].
24.
Guyre PM, Graziano RF, Vance BA, Morganelli PM, Fanger MW.
Monoclonal antibodies that bind to distinct epitopes on Fc 25. Cobbold SP, Martin G, Waldmann H. The induction of skingraft tolerance in MHC-mismatched or primed recipients: primed T-cells can be tolerized in the periphery with CD4 and CD8 antibodies. Eur J Immunol. 1990;20:2747-2755[Medline] [Order article via Infotrieve]. 26. Glennie MJ, Tutt AL, Greenman J. Preparation of multispecific F(ab)2 and F(ab)3 antibody derivatives. In: Gallagher G,Rees RC,Reynolds CW, eds. Tumor Immunobiology, a Practical Approach. Oxford: Oxford University Press; 1993:225-244. 27. Glennie M J, McBride HM, Worth AT, Stevenson GT. Preparation and performance of bispecific F(ab')2 antibody containing thioether-linked Fab' fragments. J Immunol. 1987;139:2367-2375[Abstract].
28.
Repp R, Valerius T, Sendler A, et al.
Neutrophils express the high affinity receptor for IgG (Fc 29. Peto R. Editorial: guidelines on the analysis of tumor rates and death rates in experimental animals. Br J Cancer. 1974;29:101-105[Medline] [Order article via Infotrieve]. 30. Torres RM, Law CL, Santosargumedo L, et al. Identification and characterisation of the murine homolog CD22, a lymphocyte B-restricted adhesion molecule. J Immunol. 1992;149:2641-2649[Abstract].
31.
Elsasser D, Valerius T, Repp R, et al.
HLA class II as potential target antigen on malignant B cells for therapy with bispecific antibodies in combination with granulocyte colony-stimulating factor.
Blood.
1996;87:3803-3812 32. Honeychurch J, Cruise A, Tutt AL, Glennie MJ. Bispecific Ab therapy of B-cell lymphoma: target cell specificity of antibody derivatives appears critical in determining therapeutic outcome. Cancer Immunol Immunother. 1997;45:171-173[Medline] [Order article via Infotrieve].
33.
Demanet C, Brissinck J, De Jonge J, Thielemans K.
Bispecific antibody-mediated immunotherapy of the BCL1 lymphoma: increased efficacy with multiple injections and CD28 induced co-stimulation.
Blood.
1996;87:4390-4398 34. Grafton G, Goodall M, Gregory CD, Gordon J. Mechanisms of antigen receptor dependent apoptosis of human B lymphoma cells probed with a panel of 27 monoclonal antibodies. Cell Immunol. 1997;182:45-56[Medline] [Order article via Infotrieve].
35.
Glennie MJ, McBride HM, Stirpe F, Thorpe PE, Worth AT, Stevenson GT.
Emergence of immunoglobulin variants following treatment of a B-cell leukemia with an immunotoxin composed of anti-idiotype and saporin.
J Exp Med.
1987;166:43-62
36.
Caspar CB, Levy S, Levy R.
Idiotype vaccines for non-Hodgkin's lymphoma induce polyclonal immune responses that cover mutated tumor idiotypes: comparison of different vaccine formulation.
Blood.
1997;90:3699-3706 37. Matsumoto Y, Saiki I, Murata J, Okuyama H, Tamura M, Azuma I. Recombinant human granulocyte colony-stimulating factor inhibits the metastasis of hematogenous and non-hematogenous tumors in mice. Int J Cancer. 1991;49:444-449[Medline] [Order article via Infotrieve].
38.
Davis TA, Maloney DG, Czerwinski DK, Liles TM, Levy R.
Anti-idiotype antibodies can induce long-term complete remissions in non-Hodgkin's lymphoma without eradicating the malignant clone.
Blood.
1998;92:1184-1190 39. Demanet C, Brissinck J, van Mechelen M, Leo O, Thielemans K. Treatment of murine B cell lymphoma with bispecific monoclonal antibody (anti-idiotype × anti-CD3). J Immunol. 1991;147:1091-1097[Abstract]. 40. Cragg MS, Zhang L, French RR, Glennie MJ. Analysis of the interaction of monoclonal antibodies with surface IgM on neoplastic B-cells. Br J Cancer. 1999;79:850-857[Medline] [Order article via Infotrieve].
41.
Marches R, Scheuermann RH, Uhr JW.
Cancer dormancy: role of cyclin dependent kinase inhibitors in induction of cell cycle arrest mediated via membrane IgM.
Cancer Res.
1998;58:691-697
42.
Shan D, Ledbetter JA, Press OW.
Apoptosis of malignant human B cells by ligation of CD20 with monoclonal antibodies.
Blood.
1998;91:1644-1652
43.
van Vugt M, Kleijmeer MJ, Keler T, et al.
The Fc
44.
Bennett SRM, Carbone FR, Karamalis F, Miller JFAP, Heath WR.
Induction of a CD8+ cytotoxic T lymphocyte response by cross-priming requires cognate CD4+ T cell help.
J Exp Med.
1997;186:65-70 45. Albert AL, Sauter B, Bhardwaj N. Dendritic cells acquire antigen from apoptotic cells and induce class I-restricted CTLs. Nature. 1998;392:86-89[Medline] [Order article via Infotrieve]. 46. Glennie MJ, Stevenson GT. Univalent antibodies kill tumor cells in vitro and in vivo. Nature. 1981;295:712-714. 47. Zhang L, French RR, Chan HTC, et al. The development of anti-CD79 monoclonal antibodies for treatment of B-cell neoplastic disease. Ther Immunol. 1995;2:191-202[Medline] [Order article via Infotrieve].
© 2000 by The American Society of Hematology.
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|
|
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|
 B. Stockmeyer, T. Beyer, W. Neuhuber, R. Repp, J. R. Kalden, T. Valerius, and M. Herrmann Polymorphonuclear Granulocytes Induce Antibody-Dependent Apoptosis in Human Breast Cancer Cells J. Immunol., November 15, 2003; 171(10): 5124 - 5129. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
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|
|
|
|
 |
|
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|
|
|
|
|
|
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|
|
|
|
|
|
P. Borchmann, R. Schnell, I. Fuss, O. Manzke, T. Davis, L. D. Lewis, D. Behnke, C. Wickenhauser, P. Schiller, V. Diehl, et al. Phase 1 trial of the novel bispecific molecule H22xKi-4 in patients with refractory Hodgkin lymphoma Blood, October 16, 2002; 100(9): 3101 - 3107. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
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|
|
|
|
|
|
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|
|
|
|
|
|
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|
|
|
|
|
|
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| Copyright © 2000 by American Society of Hematology Online ISSN: 1528-0020 | |||||||||
RI/CD64-directed
bispecific antibodies in B-cell lymphoma
Top
Abstract
Introduction
RI (CD89), and
Fc
background release)/(maximum
release 
, nonTg 
, nonTg +G-CSF; 
, Tg 
, Tg +G-CSF.
), anti-huCD64
F(ab')2 (
), anti-Id F(ab')2 (
),
[huCD64 × Id] (
). Symbol labeling on plots shows antibody
specificity. Anti-Id Ab preparations were selected for reaction with
A31 or BCL1 Id Ig, as appropriate. Survival was recorded
daily. Only cohorts treated with [huCD64 × Id] BsAb showed a
significant increase in survival over all control groups
(P < .02).
); [huCD64 × MHC II] (
), a
mixture of anti-CD4 and anti-CD8 + [huCD64 × Id] (
.01).
a goal that has not
been reached with anti-Id IgG mAb.
,