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Prepublished online as a Blood First Edition Paper on August 8, 2002; DOI 10.1182/blood-2002-03-0687.
IMMUNOBIOLOGY
From the Division of Hematology/Oncology, Department of
Medicine III, University Erlangen-Nürnberg, Erlangen,
Germany; the Department of Immunology, University Medical
Center Utrecht, The Netherlands; Genmab, Utrecht, The
Netherlands; and Tenovus Research Laboratory, Southampton,
United Kingdom.
Antibodies against human leukocyte antigen (HLA) class II,
such as 1D10 or Lym-1, are currently being evaluated for the treatment of B-cell lymphomas. Previous studies have demonstrated that, in
addition to IgG Fc receptors, the human myeloid IgA receptor (Fc Over the last 5 years, monoclonal antibodies have
broadened the therapeutic armentarium in oncology.1,2
Hematologic malignancies are recognized as particularly promising
targets,3 which is also reflected by today's list of
approved antibodies.4 Among those investigated in early
clinical trials against B-cell lymphomas are antibodies against human
leukocyte antigen (HLA) class II, such as Lym-15
and Hu1D10.6 Preclinical studies demonstrated that HLA
class II antibodies were particularly effective in killing B-lymphoma cells. For example, they inhibited B-cell proliferation, triggered apoptosis and complement-dependent cytotoxicity (CDC), and mediated antibody-dependent cellular cytotoxicity (ADCC) by mononuclear and polymorphonuclear neutrophil (PMN) effector
cells.7-10 Importantly, studies against xenogenic tumors
in severe combined immunodeficient (SCID) mice confirmed the
potential of HLA class II antibodies in vivo,11,12
although HLA class II antibodies were less efficient than anti-idiotype
antibodies in syngenic lymphoma models.13 However, HLA
class II is constitutively expressed on normal B cells,
monocytes/macrophages, and dendritic cells, and exposure to
interferon (IFN)- Our understanding of what effector mechanisms operate with monoclonal
antibodies in vivo is comparatively limited.16 Novel studies demonstrated that some antibodies act by directly stimulating immune effector cells17-20 or by blocking inhibitory
immune interactions.21 Traditionally, however, effector
mechanisms were divided into direct mechanisms, which are mediated by
the antibody's variable region In addition to human IgG1, antibodies of human IgA isotype were also
demonstrated to effectively trigger effector cell-mediated lysis of
solid and lymphoma tumor cells.26,27 IgA represents the
most abundantly produced antibody isotype in humans and is critically
involved in the host defense at mucosal surfaces.28 Two
isoforms Experiments reported here were approved by the Ethical Committee
of the University of Erlangen-Nürnberg (Germany), in accordance with the Declaration of Helsinki.
Monoclonal antibodies and antibody constructs
Cloning of chimeric antibodies
Production of chimeric antibodies Heavy and light chain pNUT expression vectors were cotransfected into BHK-21 cells by calcium phosphate precipitation. Transfected cells were selected in 10 µM methotrexate (Sigma, St Louis, MO). For antibody production, single cell clones were generated by limiting dilution, and cells were grown in Iscove Modified Dulbecco Medium (IMDM) supplemented with 10% heat-inactivated fetal calf serum (FCS; both Invitrogen) and 100 µM ZnCl2. For functional experiments, transfectoma supernatants were adjusted to give the indicated chimeric antibody concentrations.Immunoblotting Chimeric antibodies were run on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) with gradient gels (4%-15%), using the Phast system (Amersham Pharmacia, Buckinghamshire, United Kingdom) under reducing and nonreducing conditions. After transfer onto nitrocellulose membranes (Amersham Pharmacia), membranes were washed with distilled water and blocked for 1 hour with 5% fat-free milk in phosphate-buffered saline (PBS) containing 0.1% Tween. Immunoblots were incubated at 4°C overnight with horseradish peroxidase-conjugated polyclonal goat antihuman -light chain antibody (diluted at 1:3000) and antihuman IgG
(diluted at 1:2000; both from Serotec, Oxford, United Kingdom) or
rabbit antihuman IgA (diluted at 1:2500; Dako, Glostrup, Denmark).
After extensive washing with PBS containing 0.1% Tween, blots were
detected with the enhanced chemoluminescent reaction reagent ECL Plus
(Amersham Pharmacia).
ELISA Concentrations of chimeric antibodies were determined by sandwich ELISA, using IDEC-C2B8 (mouse/human chimeric IgG1;
Hoffmann-La Roche, Basel, Switzerland) or human polyclonal serum IgA
(Sigma) as standards. Briefly, Maxisorp ELISA plates (Nunc, Roskilde, Denmark) were coated with 2 µg/mL of polyclonal rabbit antihuman IgA
or 1 µg/mL of rabbit antihuman IgG (both from Dako). Subsequently, serial dilutions of standards and samples were added. For detection, horseradish peroxidase-conjugated goat antihuman -light chain antibody (Serotec) was used. Reactions were developed with
O-phenylenediamine dihydrochloride with urea hydrogen peroxide in
phosphate-citrate buffer (Sigma), measuring optical density
(OD) values at 450 nm. Antibody concentrations were calculated
from the standard curves.
Immunofluoresence analyses For indirect immunofluorescence, 50 µL of transfectoma supernatants were added to target cells. After washing 3 times in phosphate-buffered saline (PBS) supplemented with 1% bovine serum albumin (Sigma), 1 µg of mouse antihuman-light chain, IgA or IgG
(all from Biotest, Denville, NJ), isotype-specific mouse antihuman
IgG1, IgG2, IgG3, IgG4 (all from CLB, Amsterdam, NL), IgA1 or IgA2
(both from Southern Biotechnology, Birmingham, AL) was added.
Cells were washed again and stained with fluorescein
isothiocyanate (FITC)-labeled F(ab')2 fragments
of polyclonal goat antimouse antibodies (Jackson Immunoresearch, West
Grove, PA). After washing again, cells were analyzed on a flow
cytometer (Coulter EPICS Profile, Brea, CA). For each cell population,
relative fluorescence intensity was calculated as the ratio of mean
linear fluorescence intensity of relevant to irrelevant
isotype-matched antibodies.
Tumor cell targets Malignant B-cell lines RAJI (Burkitt lymphoma), and ARH-77 (mature B cells) were from the ATCC. Cells were kept in RF10+ medium consisting of RPMI 1640 supplemented with 10% heat-inactivated fetal calf serum (FCS), 100 U/mL penicillin, 100 U/mL streptomycin, and 4 mM L-glutamine (all from Invitrogen). For analysis of freshly isolated tumor cells, citrate-anticoagulated blood from patients with CD5/CD19-positive B-chronic lymphocytic leukemia (B-CLL) were layered over a Ficoll (Biotech, Dreieich, Germany) gradient. After centrifugation, mononuclear cells were isolated from the interface. Remaining erythrocytes were removed by hypotonic lysis. Viability of cells was tested by trypan blue exclusion. Generation of human HLA class II-transfected L66 mouse fibroblasts was described in Würflein et al.37Isolation of mononuclear and neutrophil effector cells After informed consent was obtained, 10 to 20 mL of peripheral blood was drawn from healthy volunteers or from patients receiving recombinant human granulocyte colony-stimulating factor (rhG-CSF; Neupogen, 3 to 5 µg/kg of body weight, Hoffmann La-Roche) based on clinical indications. For analysis during growth factor treatment, patients had at least 3 days of cytokine therapy. Neutrophils were isolated by a method slightly modified from that described in Elsässer et al.9 Briefly, citrate-anticoagulated blood was layered over a discontinuous Percoll (Biochrom, Berlin, Germany) gradient consisting of 70% and 62% Percoll. After centrifugation, neutrophils were collected at the interface between the 2 percoll layers and mononuclear cells were collected from the serum/Percoll interface. Remaining erythrocytes were removed by hypotonic lysis. Purity of neutrophils was determined by cytospin preparations and exceeded 95%. Mononuclear cells (MNCs) typically contained approximately 60% CD3+ T cells, 20% CD56+ natural killer (NK) cells, and only 10% CD14-expressing monocytes, as determined by immunofluorescence. Viability of cells tested by trypan blue exclusion was higher than 95%.Antibody-dependent cellular cytotoxicity assays ADCC assays were performed as described in Elsässer et al.9 Briefly, target cells were labeled with 200 µCi (7.4 MBq) 51Cr for 2 hours. After extensive washing with RF10+, cells were adjusted to 105/mL. Whole blood, plasma, or isolated effector cells (50 µL), sensitizing antibodies at a concentration of 1 µg/mL (unless otherwise indicated), and RF10+ were added to round-bottomed microtiter plates (Nunc). Assays were started by adding target cells (50 µL), resulting in a final volume of 200 µL and an effector-to-target (E/T) cell ratio of 40:1 with isolated effector cells, unless otherwise indicated. After 3 hours at 37°C, assays were stopped by centrifugation, and 51Cr release from triplicates was measured in counts per minute (cpm). Percentage of cellular cytotoxicity was calculated with the following formula: % specific lysis = (experimental cpm basal cpm )/ (maximal
cpm basal cpm) × 100, with maximal 51Cr
release determined by adding perchloric acid (3% final concentration) to target cells and basal release measured in the absence of
sensitizing antibodies and effector cells. Antibody-independent
cytotoxicity (effectors without target antibodies) was observed in
whole blood assays and with mononuclear effector cells, but not
with PMNs.
For analysis of effects induced by Fc-receptor blockade, antibodies 197 (blocking Fc C5-deficient human serum (Sigma) was used to address the role of complement in human IgG1-mediated lymphoma cell killing. Statistical analyses Group data are reported as mean ± SEM. Differences between groups were analyzed by unpaired (or, when appropriate, paired) Student t tests. Significance was accepted when the P value was less than .05.
Generation of chimeric antibodies Variable regions of murine pan-HLA class II antibody F3.3 were cloned from the hybridoma9 and sequenced (GenBank accession numbers AY058910 [VL] and AY058911 [VH]). cDNA for the light chain variable region was joined with a human constant region, and the heavy chain region
with human  1, 2,  1, 2, 3, or 4 heavy chain constant
regions, respectively. VL- and VH
heavy chain vectors were cotransfected into baby hamster kidney (BHK) cells, and stably transfected single cell clones were selected by
limiting dilution. Sandwich ELISAs served to measure antibody concentrations in supernatants, which were in the range of 5 µg/mL (Figure 1). The design of the
ELISA additionallyconfirmed pairing of heavy and light
chains.
Characterization of antibodies Chimeric antibodies of all isotypes were analyzed by SDS-PAGE. Under nonreducing conditions (Figure 2A), a single band of approximately 150 to 160 kDa was detected, corresponding to the expected size of monomeric IgG or IgA. For IgA2, an additional band of approximately 50 kDa was seen, which probably represented light chain homodimers. In the IgA2m1 isoform, which was used in our experiments, light chains form disulfide bonds with each other but not with their respective heavy chains.38 Under reducing conditions, all isotypes demonstrated single bands of approximately 50 to 60 kDa for the heavy chains and 25 kDa for the light chains (Figure 2B).
Binding characteristics of chimeric antibodies Binding of chimeric antibodies to HLA class II-transfected L66 cells was investigated by indirect immunofluorescence using FITC-labeled antihuman-light chain antibody. As shown for chimeric IgG1, IgA1, and IgA2, all isotypes demonstrated virtually identical binding at saturating concentrations (Figure
3A). Dilution experiments, furthermore,
confirmed that half-maximal binding was achieved at similar antibody
concentrations (Figure 3B). These data suggested that binding
affinities of our HLA class II antibodies were similar,39 but we cannot exclude differences in Kon or
Koff, which may be affected by antibody constant
regions.40 IgG1-4 and IgA1 or IgA2 isotype identity was
confirmed with isotype-specific secondary antibodies (Table
1). Thus, chimeric antibodies of all
isotypes demonstrated correct heavy and light chain assembly and
similar binding characteristics, which was important for subsequent
functional studies.
Comparison of chimeric antibodies in ADCC For functional studies, HLA class II-positive mature B-cell line ARH-77 was used as target cell line in 3-hour 51Cr release assays. Relative fluorescence intensity for HLA class II was 191.5 ± 28.4. All isotypes of chimeric HLA class II antibodies (IgA1, IgA2, IgG1-4) were compared in their capacity to mediate ADCC. Unseparated blood served as effector source, which was then fractionated into polymorphonuclear or mononuclear effector cells or into complement-containing plasma to identify effector mechanisms of chimeric antibodies. In unseparated blood, highest killing levels were observed with the chimeric IgG1 antibody, which was predominantly mediated by plasma but to which mononuclear and PMN effector cells also contributed (Figure 4). Plasma-mediated killing was completely abrogated when plasma was heat-inactivated (30 minutes at 56°C) or when C5-deficient human serum was used, indicating that lysis was complement dependent (data not shown). Considerably lower tumor cell killing was observed with other IgG isotypes. Interestingly, human IgA1 and IgA2 also effectively triggered lymphoma cell lysis. Analyses of effector mechanisms demonstrated that IgA-mediated killing was exclusively triggered by PMNs. When RAJI Burkitt lymphoma cells were used as targets (relative fluorescence intensity [RFI] for HLA class II expression, 134.8 ± 21.6) in ADCC by isolated PMNs, IgG1 antibodies proved to be even less effective. Significant killing was observed only at high effector-to-target ratios. In contrast, IgA-mediated lysis of RAJI cells by isolated PMNs was comparable to results with ARH-77. Notably, significant ADCC was observed over a broad range of effector-to-target ratios (Figure 5A) and antibody concentrations (Figure 5B).
Fc receptor involvement in ADCC by chimeric IgA antibodies Involvement of Fc receptors in ADCC mediated by chimeric IgA1 and IgA2 was investigated using blocking antibodies to FcRI (CD89),
FcRI (CD64), FcRII (CD32), or FcRIII (CD16). With PMN effector
cells, blocking FcRI resulted in complete inhibition of lysis
(98.3% ± 1.4% or 95.2% ± 2.9%, n = 4), while blockade of
Fc receptors did not significantly affect IgA-induced ADCC (Figure
6).
Killing of primary B-CLL cells by chimeric HLA class II antibodies Primary tumor cells are generally more difficult to kill than lymphoma cell lines. Therefore, chimeric IgG1, IgA1, or IgA2 versions of HLA class II antibody F3.3 were compared in ADCC against freshly isolated B-CLL cells. As effector source, unseparated blood from healthy volunteers or from G-CSF-primed patients was used (Figure 7). G-CSF-primed blood contained significantly higher PMN numbers than healthy donor blood (29 000 ± 7000/µL vs 2900 ± 500/µL, n = 4), while FcRI
expression was not affected by G-CSF application.41 Both
donor groups mediated significant killing with all 3 antibody isotypes.
Analyses of effector mechanisms confirmed results obtained with cell
lines: IgG1 killed via complement and MNCs, while IgA acted exclusively
via PMNs (data not shown). Notably, both IgA1- and IgA2-mediated lysis
were significantly (P < .05) enhanced in blood from
donors undergoing G-CSF therapy compared with healthy
donors.
As demonstrated in this study, recombinant chimeric IgA
isoforms of pan-HLA class II antibody F3.3 proved highly effective in
killing human lymphoma cell lines and, more important, primary tumor
cell isolates from CLL patients. Notably, the killing mechanisms of IgA
and IgG1 antibodies differed significantly. While IgG1 effectively
triggered complement-dependent lysis and ADCC by mononuclear effector
cells, IgA antibodies acted exclusively via ADCC by PMNs. Although
monocytes also express CD89 and were reported to kill via
Fc In our experiments, IgA-mediated killing by PMNs was completely blocked
by Fc Today, human IgG1 is the most widely used antibody isotype in oncology,
because it effectively triggers NK cell-mediated ADCC and CDC and Application of IgA antibodies may be another, or a complementary
approach, to improve effector cell recruitment for antibody therapy. As
demonstrated here, IgA antibodies would be expected to effectively
recruit PMNs, the most populous effector cell in vivo, which can be
further expanded and activated by application of G-CSF or GM-CSF.
However, PMNs' capacity to trigger tumor cell lysis is strongly
dependent on the selection of appropriate tumor target
antigens.9,37,52 Experiments with chimeric HER-2/neu and
CD19 antigens, in which intra- and extracellular domains were exchanged, demonstrated that intracellular domains of target antigens were critical for PMN-mediated killing.53 Further studies
are required to analyze whether IgA antibodies may overcome PMNs' "antigen restriction," as was partially observed with
Fc
We thank S. Gehr, B. Bock, and H. Vile for their expert technical assistance.
Submitted March 6, 2002; accepted July 22, 2002.
Prepublished online as Blood First Edition Paper, August 8, 2002; DOI 10.1182/blood-2002-03-0687.
Supported by the ELAN Fonds from the University Erlangen-Nürnberg, by the Wilhelm Sander Stiftung, and by a Research Training Grant from the European Community Biotechnology program (BIO4-CT97-5084 to G.V.).
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: Thomas Valerius, Division of Hematology/Oncology, Department of Medicine III, University of Erlangen-Nürnberg, Krankenhausstr 12, 91054 Erlangen, Germany; e-mail: thomas.valerius{at}med3.imed.uni-erlangen.de.
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Top
Abstract
Introduction
1 affinity to the
myeloid IgA receptor Fc
