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Blood, Vol. 93 No. 11 (June 1), 1999:
pp. 3922-3930
Humanized Anti-HM1.24 Antibody Mediates Myeloma Cell Cytotoxicity
That Is Enhanced by Cytokine Stimulation of Effector Cells
By
Shuji Ozaki,
Masaaki Kosaka,
Yuji Wakahara,
Yasuko Ozaki,
Masayuki Tsuchiya,
Yasuo Koishihara,
Tetsuya Goto, and
Toshio Matsumoto
From the First Department of Internal Medicine, School of Medicine,
University of Tokushima, Tokushima, and Fuji-Gotemba Research
Laboratory, Chugai Pharmaceutical Co, Ltd, Shizuoka, Japan.
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ABSTRACT |
To develop a new immunotherapy for multiple myeloma, we have
generated a monoclonal antibody (MoAb) that detects a human plasma cell-specific antigen, HM1.24. Our previous study has shown that mouse
anti-HM1.24 MoAb inhibits the proliferation of human myeloma cells
implanted into severe combined immunodeficiency mice. In this report,
we evaluated the antitumor activity of the humanized anti-HM1.24 MoAb
(IgG1 ), which was constructed by grafting the complementarity-determining regions. In contrast to the parent mouse
MoAb, humanized anti-HM1.24 MoAb mediated antibody-dependent cellular
cytotoxicity (ADCC) against both myeloma cell lines and myeloma cells
from patients in the presence of human peripheral blood mononuclear
cells (PBMCs). The PBMCs from untreated myeloma patients exhibited ADCC
activity as efficiently as those of healthy donors. Although decreased
ADCC activity of PBMCs was observed in patients who responded poorly to
conventional chemotherapy, it could be significantly augmented by the
stimulation with interleukin-2 (IL-2), IL-12, or IL-15. There was a
strong correlation between the percentage of CD16+ cells
and ADCC activity in the PBMCs of myeloma patients. Moreover, peripheral blood stem cell collections from myeloma patients contained higher numbers of CD16+ cells than PBMCs and exhibited
ADCC activity that was enhanced by IL-2. These results indicate that
humanized anti-HM1.24 MoAb has potential as a new therapeutic strategy
in multiple myeloma and that treatment of effector cells with
immunomodulating cytokines can restore the effect of humanized
anti-HM1.24 MoAb in patients with diminished ADCC activity.
© 1999 by The American Society of Hematology.
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INTRODUCTION |
MULTIPLE MYELOMA remains an incurable
malignancy despite certain advances in chemotherapeutic regimens.
Conventional chemotherapy results in a low complete response rate and
disease progression usually occurs within a few years.1,2
Although myeloablative chemotherapy followed by allogeneic or
autologous hematopoietic stem cell transplantation has increased the
incidence of complete remission, relapses are still
observed.3-5 Thus, new forms of maintenance chemotherapy
and/or immunotherapy are needed to eliminate minimal residual disease
and improve prognosis.
Antibody-dependent cellular cytotoxicity (ADCC) is one of the most
important weapons of the immune response against tumor cells.6,7 This activity is mediated by tumor-specific
antibodies and Fc receptor-bearing effector cells, such as natural
killer (NK) cells, T lymphocytes, and phagocytes.8,9
Importantly, ADCC depends on the cytolytic activity of these effector
cells. However, because ADCC activity and/or NK cell function are
suppressed in certain cancer patients,9,10 this
immunotherapeutic approach has not proved as successful as originally
expected. Nevertheless, we and others have observed an increased number
and activity of NK cells in the peripheral blood and bone marrow of
myeloma patients.11-14 These cells could act as effector
cells to kill antibody-coated target cells by an ADCC mechanism.
Immunotherapy with tumor cell-specific antibodies, therefore, might be
a valuable treatment option for myeloma patients.
Although several researchers have developed plasma cell-specific
antibodies,15-20 the application of these antibodies to the immunotherapy of multiple myeloma has not been extensively
investigated. Recently, we have generated a mouse monoclonal antibody
(MoAb) to a novel plasma cell-specific antigen, termed HM1.24, for the treatment of multiple myeloma.21 HM1.24 is a type II
transmembrane protein that has a molecular weight of 29 to 33 kD22 and is expressed selectively on
terminally differentiated normal and neoplastic B cells.21
Our previous studies have shown that anti-HM1.24 MoAb accumulates in
human myeloma xenografts in severe combined immunodeficiency (SCID)
mice and induces strong antitumor activity by an ADCC
mechanism.23-25 Thus, HM1.24 antigen is an attractive target for the immunotherapy of multiple myeloma and its biological function in normal and myeloma cells is under investigation.
To develop an immunotherapeutic agent for clinical use, we have
constructed humanized anti-HM1.24 MoAb (IgG1) by grafting the
complementarity-determining regions from the parent mouse MoAb to a
human MoAb. In this study, we evaluate the antitumor activity of
humanized anti-HM1.24 MoAb and the cytolytic activity of various
effector cells from myeloma patients. Our results indicate that
humanized anti-HM1.24 MoAb can mediate ADCC activity against myeloma
cells in the presence of effector cells of patients, providing supportive data for clinical trials of immunotherapy for multiple myeloma and related plasma cell dyscrasias.
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MATERIALS AND METHODS |
Patients.
The diagnosis and clinical staging of multiple myeloma were performed
according to the criteria of Durie and Salmon.26 A total of
45 treated or untreated myeloma patients were included in this study.
Their mean age was 65.3 years (range, 40 to 96), with 21 males and 24 females. Clinical stage was distributed as follows: IA, 22%; IIA,
24%; IIIA, 49%; and IIIB, 5%. Monoclonal serum or urine Ig was found
in 98% of patients: IgG 62%; IgA, 22%; IgD, 7%; and light chain
only, 7%. The / light chain isotype ratio was 1.4.
Patients were treated with melphalan-prednisone with or without
vincristine and anthracyclins. For those patients receiving intermittent chemotherapy, samples were taken at least 4 weeks after
the last cycle of chemotherapy. In five patients undergoing autologous
peripheral blood stem cell transplantation (PBSCT), PBSCs were
mobilized by an intravenous injection of 3 g/m2
cyclophosphamide on days 1 and 2 followed by daily subcutaneous administration of 5 µg/kg granulocyte colony-stimulating factor (G-CSF). Three patients received PBSCT and their blood samples were
examined 2, 3, or 9 months after PBSCT. Response to treatment was
assessed following the criteria defined by Gore et al.27 Complete response was defined by the disappearance of the paraprotein (evaluated by immunoelectrophoresis) from the serum and the
concentrated urine and less than 5% plasma cells in the bone marrow.
Patients were considered to be in partial response when a decrease of
more than 50% was observed in measurable paraprotein and bone marrow infiltration. All others were regarded as nonresponders.
Preparation of effector cells.
Peripheral blood or bone marrow samples were obtained from healthy
donors or patients with multiple myeloma after informed consent.
Peripheral blood mononuclear cells (PBMCs) or bone marrow mononuclear
cells (BMMCs) were separated by Ficoll-Conray (density, 1.077) gradient
centrifugation. Polymorphprep (density, 1.113) gradient
(Nycomed Pharma AS, Oslo, Norway) was used to purify neutrophils. PBSCs
were collected by a Fenwal CS-3000 Plus cell separator (Baxter,
Deerfield, IL) during the phase of bone marrow recovery after
chemotherapy as described above. PBSCs were separated by 40% and 60%
Percoll gradients (Pharmacia, Uppsala, Sweden).
Antibodies.
Mouse anti-HM1.24 MoAb (IgG2a) was produced by the fusion of mouse
myeloma cells SP2/0 with spleen cells from Balb/c mice immunized with
the human myeloma cell line, KPC-32.21 This MoAb recognizes
a 29- to 33-kD glycoprotein as shown by immunoprecipitation assay under
reducing conditions. The anti-HM1.24 MoAb was purified from the ascites
fluid by ammonium sulfate precipitation and a protein A-affinity
chromatography kit (Ampure PA; Amersham Japan, Tokyo, Japan). The
mouse-human chimeric anti-HM1.24 MoAb was constructed by linking the
cDNA sequences encoding the heavy and light chain variable regions of
mouse anti-HM1.24 with the cDNAs encoding the human  1 and constant regions, respectively,28
using human elongation factor (HEF)
expression vectors.29 Humanized anti-HM1.24 MoAb (IgG1)
was constructed by grafting the complementarity-determining regions
from the mouse MoAb to a human MoAb.28 A more
detailed description of this procedure has been provided
previously.30 Mouse IgG2a (UPC-10; Cappel, Malvern, PA) and
human IgG1 (Serotec, Oxford, UK) proteins were used as control IgG.
For inhibition studies, heat-aggregated human IgG was prepared by a
20-minute incubation at 63°C.
Myeloma cells.
Myeloma cells were obtained from the bone marrow of a patient (no. 1)
and from the malignant pleural effusion of three patients (no. 2, no.
3, and no. 4). Mononuclear cells were isolated by Ficoll-Conray density
gradient centrifugation, and adherent cells and T cells were depleted
as described.31 The mononuclear cell fraction of these
samples included more than 95% CD38+ myeloma cells and was
used for the target of ADCC assay.
The human plasma cell lines, U266 and ARH-77, were obtained from the
American Type Culture Collection (Rockville, MD). The following cell
lines were obtained from the Japanese Cancer Research Resources Bank
(Tokyo, Japan): RPMI 8226, IM-9, HS-Sultan, Ramos, Daudi, and HEL.
Myeloid HEL cells which do not express the HM1.24 antigen were used as
control cells. These cells were cultured in RPMI 1640 medium
supplemented with 10% fetal bovine serum, 100 U/mL penicillin, and 100 µg/mL streptomycin.
Construction of HM1.24 expressing CHO cells.
To establish stable Chinese hamster ovary (CHO) transformants
expressing the HM1.24 antigen, cDNA was transfected into CHO cells and
transformants were selected in the presence of 500 µg/mL G418.
Transformants that expressed different amounts of HM1.24 were obtained
and used for the targets of ADCC assay.
Flow cytometry.
Expression of HM1.24 antigen on myeloma cells was examined by flow
cytometry. Cells were washed with cold phosphate-buffered saline (PBS)
and stained on ice for 30 minutes with mouse anti-HM1.24 MoAb or
control IgG. After incubation with primary MoAb, cells were washed
three times with cold PBS containing 0.1% bovine serum albumin and
0.02% sodium azide and then incubated on ice for an additional 30 minutes with fluorescein isothiocyanate (FITC)-conjugated goat
F(ab')2 anti-mouse IgG antibody (Tago, Burlingame, CA). The cells were washed again and resuspended in 1% paraformaldehyde. In
some experiments, cells were stained with biotin-labeled isotype control IgG or anti-HM1.24 MoAb, and phycoerythrin (PE)-conjugated anti-CD38 MoAb (Becton Dickinson, San Jose, CA), and then with streptavidin-RED 670 (GIBCO-BRL, Rockville, MD). The analysis was
performed on a flow cytometer (EPICS XL; Coulter Electronics, Hialeah,
FL). The mean specific fluorescence intensity (MSFI) was calculated as
the ratio of mean fluorescence channel of anti-HM1.24 MoAb/control MoAb.
To analyze the HM1.24 expression on hematopoietic progenitor cells in
PBSC collections, cells were stained with FITC-conjugated control IgG
or mouse anti-HM1.24 MoAb, and PE-conjugated anti-CD34 MoAb
(PharMingen, San Diego, CA). The CD34+ fraction was gated
based on CD34 fluorescence intensity and side scatter profile. To
examine the phenotype of effector cells from patients, cells were
stained with FITC-labeled anti-CD3, anti-CD4, anti-CD8, anti-CD16,
anti-CD20, or PE-labeled anti-CD56 MoAbs (Becton Dickinson) and were
analyzed as above.
Complement-dependent cytotoxicity.
Cell lysis with complement was determined using a
51Cr-release assay. Target cells were labeled with 0.1 mCi
51Cr-sodium chromate (New England Nuclear, Boston, MA) at
37°C for 1 hour. The cells were then washed three times with RPMI
1640 medium. 51Cr-labeled cells (1 × 104
cells) were incubated with various concentrations of anti-HM1.24 MoAb
or control IgG on ice for 30 minutes. The unbound antibody was removed
by washing the cells three times with medium. The cells were then
distributed into 96-well plates and incubated with serial dilutions of
baby rabbit complement (Cedarlane, Ontario, Canada) or human serum at
37°C for 2 hours. After incubation, supernatants from each well (50 µL) were harvested and 51Cr was measured using a gamma
counter. Spontaneous release of 51Cr was measured after
incubating 51Cr-labeled cells with medium alone. The
maximum release of 51Cr was determined after incubation of
51Cr-labeled cells with 1% NP-40. Percentage of
cytotoxicity was calculated from the formula: specific cytotoxicity
(%) = (A  C)/(B C) × 100, where A = experimental
51Cr release, B = maximum 51Cr release, and C = spontaneous 51Cr release.
ADCC assay.
ADCC activity was determined by standard 4-hour
51Cr-release assay. In some experiments, effector cells
were cultured in RPMI 1640 medium with or without recombinant human
interleukin-2 (IL-2) (Genzyme, Cambridge, MA), IL-10 (Genzyme), IL-12
(R&D Systems, Minneapolis, MN), IL-15 (Genzyme) or macrophage
colony-stimulating factor (M-CSF; Morinaga Milk Industry Co, Ltd,
Tokyo, Japan) for 3 days, then washed and resuspended in medium before
use. 51Cr-labeled target myeloma cells
(1 × 104 cells) were placed in 96-well plates and
various concentrations of anti-HM1.24 MoAb or control IgG were added to
wells. Effector cells were then added to the plates at various effector
to target (E/T) ratios. After 4-hour incubation, supernatants were
removed and counted in a gamma counter. The percentage of cell lysis
was determined as above.
Hematopoietic progenitor cell assay.
The effect of humanized anti-HM1.24 MoAb on the growth of
granulocyte-macrophage colony-forming units (GM-CFU) and erythroid burst-forming units (E-BFU) was evaluated as described
previously.31 Briefly, PBSC collections
(1 × 105 cells) from myeloma patients were incubated
with 10 µg/mL control human IgG or humanized anti-HM1.24 MoAb in
Iscove's modified Dulbecco's medium (IMDM) for 30 minutes at 37°C.
The cells were then plated in 35-mm tissue-culture dishes in 1 mL of
IMDM containing 1% methylcellulose, 20% fetal calf serum, 1% bovine
serum albumin, 450 ng/mL iron-saturated transferrin, 10 ng/mL IL-3, and
2 U/mL erythropoietin or 10 ng/mL G-CSF in triplicate. After 14 days of
culture, the numbers of colonies were counted by an inverted microscope.
Statistical analysis.
The statistical significance of difference between groups was analyzed
by unpaired t-test. The correlation between ADCC activity and
HM1.24 expression on target cells or phenotypic data of PBMCs was
evaluated by Pearson's rank correlation analysis.
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RESULTS |
ADCC activity of humanized anti-HM1.24 MoAb against RPMI 8226 cells.
The ability of chimeric and humanized versions of anti-HM1.24 to
mediate ADCC was determined in a 51Cr-release assay. As
shown in Fig 1, mouse anti-HM1.24 MoAb did not induce ADCC activity
against RPMI 8226 cells in the presence of PBMCs from healthy donors,
suggesting that the mouse form of this MoAb is completely unrecognized
by effector cells. In contrast, humanized as well as chimeric
anti-HM1.24 MoAb induced ADCC activity in a dose-dependent manner (Fig
1A) and the extent of cytotoxicity was
dependent on the E/T ratio (Fig 1B). This cytotoxicity was mediated by
humanized anti-HM1.24 MoAb even at a low concentration of 0.01 µg/mL.
No additional killing was seen at concentrations above 10 µg/mL. In
contrast, neutrophils isolated from healthy donors or myeloma patients
did not exhibit ADCC activity in the presence of humanized anti-HM1.24
MoAb even after stimulation with G-CSF (data not shown).
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| Fig 1.
ADCC activity against RPMI 8226 cells by normal human
PBMCs. (A) 51Cr-labeled RPMI 8226 cells were incubated with
PBMCs at an E/T ratio of 50 along with various concentrations of
antibodies. (B) 51Cr-labeled RPMI 8226 cells were incubated
with PBMCs in the presence of 1 µg/mL of antibodies. Symbols
represent control human IgG ( ), mouse anti-HM1.24 MoAb ( ),
chimeric anti-HM1.24 MoAb ( ) and humanized anti-HM1.24 MoAb ( ).
Data represent the mean ± SD of triplicates.
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To further examine the role of the Fc region of the humanized
anti-HM1.24 MoAb, competitive inhibition studies with mouse anti-HM1.24
MoAb were performed. The humanized anti-HM1.24 MoAb-mediated cytotoxicity was inhibited by mouse anti-HM1.24 MoAb in a
dose-dependent manner, suggesting that cytotoxicity was mediated by the
Fc region of the humanized MoAb (Fig 2).
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| Fig 2.
Effects of mouse anti-HM1.24 MoAb and human IgG on ADCC
activity mediated by humanized anti-HM1.24 MoAb.
51Cr-labeled RPMI 8226 cells were incubated with PBMCs from
healthy donors (E/T ratio, 50) and humanized anti-HM1.24 MoAb (1 µg/mL) in the presence of mouse anti-HM1.24 MoAb ( ),
heat-aggregated human IgG (), or monomeric human IgG (). Effector
cells were preincubated for 15 minutes at room temperature with
monomeric or heat-aggregated IgG. Data represent the mean ± SD of
triplicates. *P < .05 or  P < .005, compared with the data in the absence of mouse anti-HM1.24 MoAb or
human IgG preparations.
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We next determined whether human IgG or paraproteins from myeloma
patients could inhibit ADCC activity by blocking the FcR of effector
cells. The preincubation of PBMCs with monomeric human IgG did not
affect the cytotoxicity mediated by humanized anti-HM1.24 MoAb, whereas
heat-aggregated human IgG inhibited the effect of humanized anti-HM1.24
MoAb in a dose-dependent manner (Fig 2). Similarly, serum from IgG or
IgA myeloma patients (serum dilution, 1:4) did not abrogate this ADCC
activity (data not shown), indicating that this humanized
MoAb mediates ADCC through FcR even in the presence of monomeric
IgG or paraproteins. These findings also suggest that soluble
HM1.24 antigen blockage of the binding of humanized anti-HM1.24 MoAb
did not occur in the serum of myeloma patients.
Complement-dependent cytotoxicity.
The complement-dependent cytotoxicity of anti-HM1.24 MoAb was examined
in the presence of rabbit or human complement. Both humanized and mouse
anti-HM1.24 MoAb (10 µg/mL) mediated the complement-dependent cytotoxicity (24.2% ± 5.7% and 67.1% ± 2.5%, mean ± SD
of triplicates, respectively) against RPMI 8226 cells with rabbit
complement (complement dilution, 1:5). However, no cytotoxicity was
observed when human serum from healthy donors or myeloma patients was
used as the source of complement. There was no complement-dependent
cytotoxicity elicited by humanized or mouse anti-HM1.24 MoAb against
HEL cells.
Expression of HM1.24 and the sensitivity to humanized anti-HM1.24
MoAb of myeloma cells.
Next, additional myeloma cell lines as well as myeloma cells from
patients were used as targets, and surface expression of HM1.24 on
these cells was examined using indirect immunofluorescence techniques.
HM1.24 antigen was strongly expressed on ARH-77, IM-9, RPMI 8226, Ramos, U266, HS-Sultan, Daudi, and CD38+ myeloma cells from
patients (Fig 3). Mean specific
fluorescence intensity (MSFI) was determined by immunofluorescence
staining, and the results are shown in Table
1. The MSFI ranged between 12 and 43 in
these myeloma cells.
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| Fig 3.
Flow cytometric analysis of HM1.24 expression on myeloma
cells. RPMI 8226 (A) and BMMCs from patient no. 1 (B) were stained with
biotin-labeled isotype control IgG or anti-HM1.24 MoAb, and
PE-conjugated anti-CD38 MoAb, and then with streptavidin-RED 670. Myeloma cell regions (R) were gated for further analysis according to
the side scatter (SS) profile and CD38 expression. BMMCs from patient
no. 1 contained 95% of CD38+ myeloma cells (B). The MSFI
was calculated as the ratio of mean fluorescence channel of anti-HM1.24
MoAb/control IgG.
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The ADCC activity of PBMCs from a healthy donor was tested against
these myeloma cells. Both myeloma cell lines and myeloma cells from
patients were killed with PBMCs in the presence of humanized
anti-HM1.24 MoAb (Table 1). There was no significant correlation
between HM1.24 expression levels and cytolysis of these myeloma cells.
However, the degree of target cell cytolysis was related to the level
of HM1.24 expression in CHO cells transfected with HM1.24 cDNA
(r = .94, P = .08). PBMCs from myeloma patients also mediated ADCC activity against autologous myeloma cells (Table 1).
Morphological examination showed that these myeloma cells were attached
to large granular lymphocytes, and cytolysis of myeloma cells was only
observed in the presence of humanized anti-HM1.24 MoAb (Fig
4). In contrast, no cytolysis of HEL cells
was seen with humanized anti-HM1.24 MoAb.
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| Fig 4.
Morphology of effector cells attacking myeloma cells.
Myeloma cells were purified from the bone marrow (patient no. 1) and
were cultured with PBMCs from a healthy donor in the presence of
humanized anti-HM1.24 MoAb (1 µg/mL) for 30 minutes. Cytospin
preparations were stained with Wright-Giemsa (original magnification × 330).
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ADCC activity of PBMCs from myeloma patients.
The ADCC activity of PBMCs from both treated and untreated myeloma
patients was examined using RPMI 8226 cells as target cells. The mean
ADCC activity of PBMCs from healthy donors was 31.2% ± 7.6%
(mean ± SD, n = 12). As shown in Fig
5, PBMCs of untreated myeloma patients had
ADCC activity as efficient as that of healthy donors despite different
clinical stages. No significant differences in ADCC activity were found
between the various clinical stages. The mean values of ADCC activity
were not significantly different in patients with different types of
paraproteins (data not shown). ADCC activity was also observed in
treated patients, including those after PBSCT (n = 3), while in three
of the stage III patients decreased ADCC activity was exhibited (less
than 2 SD of controls, <16%).
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| Fig 5.
ADCC activity of PBMCs from myeloma patients according to
clinical stages. PBMCs from healthy donors or untreated (), treated
(), or post PBSCT () myeloma patients were compared as effector
cells against RPMI 8226 cells at an E/T ratio of 50 in the presence of
1 µg/mL humanized anti-HM1.24 MoAb. Horizontal bars represent the
mean values of each group. The shaded area represents the mean ± 2 SD
of ADCC activity of PBMCs from healthy donors.
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Clinical characteristics according to ADCC activity.
To assess different ADCC activities among the treated patients in stage
III, clinical characteristics at the time of ADCC assay were evaluated.
Three patients displaying low ADCC activity (<16%, <2 SD of
controls) were nonresponders to chemotherapy with relatively severe
anemia and high levels of paraprotein. In contrast, patients with high
ADCC activity (>46%, >2 SD of controls) were all in
complete response after treatment. Although there was no significant
difference in white blood cell counts between the patients with low and
high ADCC activity, the number of CD16+ cells in the
peripheral blood was relatively lower in the group with low ADCC activity.
Correlation between the percentage of NK cells and ADCC activity in
myeloma.
Because NK cells, which express CD16 and CD56, are known to be the
major effector cells of ADCC,9 we analyzed the percentage of NK cells among the PBMCs by flow cytometry. ADCC activity of PBMCs
correlated significantly with the percentages of CD16+
cells (r = .69, P < .0001; Fig
6) or CD56+ cells (r = .56,
P = .0011) in the peripheral blood. In contrast, the
percentages of CD3, CD4, CD8, CD20, or CD19 positive cells did not
correlate with ADCC activity (data not shown).
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| Fig 6.
Correlation between ADCC activity and the percentage of
CD16+ cells in PBMCs from myeloma patients.
51Cr-labeled RPMI 8226 cells were incubated with PBMCs from
myeloma patients at an E/T ratio of 50 in the presence of 1 µg/mL
humanized anti-HM1.24 MoAb. A regression line is shown
(Y = 0.48X + 18.8).
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Effect of cytokines on ADCC activity of effector cells.
To evaluate whether cytokines can enhance the diminished ADCC activity
found in certain myeloma patients, we examined the effect of various
cytokines (IL-2, IL-10, IL-12, IL-15, or M-CSF) on the ADCC activity of
PBMCs. PBMCs were incubated for 3 days in culture medium with IL-2 (500 U/mL), IL-10 (20 ng/mL), IL-12 (20 ng/mL), IL-15 (20 ng/mL), or M-CSF
(5,000 U/mL) and then added to cultures of 51Cr-labeled
RPMI 8226 cells with humanized anti-HM1.24 MoAb (1 µg/mL).
As shown in Fig 7, the cytolytic activity
of PBMCs activated by IL-2, IL-12, or IL-15 without humanized
anti-HM1.24 MoAb, ie, lymphokine-activated killer (LAK) cell activity,
was found in both healthy donors and myeloma patients. ADCC activity
with humanized anti-HM1.24 MoAb was not augmented by the stimulation
with these cytokines in healthy donors. In contrast, reduced ADCC
activity of PBMCs from certain myeloma patients was significantly
enhanced by IL-2, IL-12, or IL-15. Cytolytic activity by ADCC was
always higher than NK or LAK activity. Although IL-10 and M-CSF have been shown to stimulate the ADCC activity of
monocytes,32,33 both cytokines failed to enhance the
cytolytic activity of PBMCs from either healthy donors or myeloma
patients.
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| Fig 7.
Effects of cytokines on ADCC activity of PBMCs against
RPMI 8226. PBMCs from healthy donors (A, n = 5) and myeloma
patients (B, n = 5) were cultured with various cytokines for 3 days
and were used as effector cells in the presence of 1 µg/mL control
human IgG () or humanized anti-HM1.24 MoAb (). Cytotoxicity was
determined by a 4-hour 51Cr-release assay at an E/T ratio
of 50. Data represent the mean ± SD of triplicates. *P < .05 or P < .001, compared with the data of nonactivated
PBMCs in the presence of humanized anti-HM1.24 MoAb.
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Effector cell analysis of ADCC in myeloma patients.
Finally, the ADCC activity of PBMCs, BMMCs, and PBSC collections from
myeloma patients was examined (Table 2).
The proportion of CD16+ cells and ADCC activity was
relatively higher in PBMCs than in BMMCs. However, despite a high
percentage of CD16+ cells in PBSC collection, the magnitude
of ADCC activity was relatively low as compared with that of PBMCs.
Again, the ADCC activity of PBSC collections as well as in PBMCs was
significantly enhanced by the stimulation with IL-2.
Effect of humanized anti-HM1.24 MoAb on hematopoietic progenitor
cells.
To determine the safety of humanized anti-HM1.24 MoAb for hematopoietic
progenitor cells, the expression of HM1.24 and the effect of humanized
anti-HM1.24 MoAb on these cells were investigated. CD34+
progenitor cells in PBSC collections did not express HM1.24 antigen by
two-color flow cytometry (data not shown). In addition, the humanized
anti-HM1.24 MoAb did not significantly inhibit the growth of GM-CFU and
E-BFU grown from PBSCs of myeloma patients (n = 5) when compared with
control IgG.
| |
DISCUSSION |
In this report, we have shown that humanized anti-HM1.24 MoAb can
mediate ADCC activity against myeloma cell lines as well as against
neoplastic myeloma cells from patients in the presence of effector
cells. These findings confirm our previous observation that HM1.24
serves as a target molecule for myeloma immunotherapy.23-25 Moreover, most myeloma patient PBMCs showed ADCC activity comparable with that of healthy donors, although a small number of PBMCs from
patients with refractory disease did show decreased ADCC activity. This
decreased ADCC activity, however, could be significantly enhanced by
treatment with IL-2, IL-12, or IL-15. Previous studies have suggested
that the PBMCs of myeloma patients have NK and LAK
activity,34,35 but little is known about ADCC specific for
myeloma cells. We have shown that various effector cells, including
PBMCs, BMMCs, and PBSC collections, have the ability to induce ADCC
activity against myeloma cells together with humanized anti-HM1.24
MoAb. Indeed, this cytotoxicity was specific and more effective than NK
or LAK activity against myeloma cells. Thus, humanized anti-HM1.24 MoAb
has therapeutic potential for myeloma patients even with advanced disease.
Among antiplasma cell antibodies previously described, only chimeric
and humanized forms of anti-CD38 have been reported to have cytotoxic
potential against myeloma cells in the presence of effector cells.
Stevenson et al18 and Ellis et al20 have shown
that the maximum ADCC activity of these chimeric and humanized anti-CD38 MoAbs was 40% to 60% in the presence of normal PBMCs. Although the target cells were different in our cytotoxicity assay, we
found that humanized anti-HM1.24 MoAb elicited ADCC activity as
efficiently as anti-CD38 MoAbs in the presence of normal PBMCs. However, CD38 is also expressed on normal tissues such as hematopoietic cells.36 In contrast, HM1.24 antigen was not expressed on
normal tissues including the peripheral blood, bone marrow, lymph node, liver, spleen, kidney, heart,21 and hematopoietic
progenitor cells, suggesting that humanized anti-HM1.24 MoAb would not
cause any adverse effects on these normal cells. Indeed, humanized
anti-HM1.24 MoAb did not show toxicity for progenitor cells in PBSC
collections, even though CD16+ effector cells were present
in the assay.
The effector cells responsible for ADCC have not been fully identified,
but several studies have indicated that human IgG1 MoAb is most
effective in cell-mediated cytolysis, utilizing FcRIII (CD16)-expressing NK cells as the effectors.37 We have
shown that both humanized and mouse-human chimeric anti-HM1.24 MoAbs that have human C1 can mediate ADCC against human myeloma cells, whereas the parent mouse MoAb has no effect. In addition, ADCC activity
of PBMCs correlated significantly with the percentages of
CD16+ cells. Moreover, heat-aggregated IgG blocked the
binding site of FcR on effector cells, but monomeric human IgG and
patient serum containing paraprotein did not inhibit the ADCC activity by humanized anti-HM1.24 MoAb in accordance with the findings that
FcRIII was not blocked by monomeric IgG.38 In contrast, neutrophils that also express Fc receptors did not exhibit any cytotoxicity with humanized anti-HM1.24 MoAb. These results indicate that Fc-mediated effector functions, especially through FcRIII, are
responsible for the antitumor activity observed, and that an increase
of NK cells that express CD16 and CD56 is a good indicator for high
ADCC activity.
Because of the wide variation in the ADCC activity of PBMCs in healthy
controls, there was no apparent suppression of ADCC activity in myeloma
patients. However, three patients with refractory disease showed
decreased ADCC activity of PBMCs (<2 SD of controls). In contrast, a
high level of ADCC activity was observed in patients in which treatment
proved successful. Similar observations have been reported that myeloma
patients in stable remission phase showed a significant recovery of LAK
activity.34 These findings suggest that cell-mediated
immunity can recover following effective chemotherapy. Further studies
will be necessary to determine whether ADCC activity is of prognostic
value in patients with multiple myeloma.
The precise mechanism of reduced ADCC activity in patients with
advanced disease remains unclear, but the presence of suppressor factors has been suggested that can alter NK cell function, regulated by several cytokines, such as IL-2, IL-12, and IL-15.39-41
Abnormal cytokine production, ie, decreased serum levels of IL-2 and
increased serum levels of IL-1 , IL-6, and prostaglandin
E2, has been reported in patients with various types of
cancer.42 Specifically, overproduction of IL-6 and
transforming growth factor- (TGF-) was observed in myeloma
patients in association with a poor prognosis.43-45 Because
IL-6 and TGF- are known to suppress the production of IL-2,
interferon-, and tumor necrosis factor from T
cells,46,47 they might negatively regulate the immune
response of NK cells. In fact, we found that decreased ADCC activity of
PBMCs can be restored by IL-2, IL-12, and IL-15 in myeloma patients,
but that these cytokines cannot enhance ADCC activity of PBMCs from
healthy donors. Although there is no direct evidence for an established role of these cytokines in cellular immunity, our data support the
hypothesis that an abnormal cytokine network might contribute to
reduced ADCC activity, at least in part, and suggest the potential benefits of ex vivo treatment with IL-2, IL-12, or IL-15 for
immunotherapy, especially in patients with advanced disease.
PBSCs are increasingly used as an alternative to autologous bone marrow
for hematological rescue after myeloablative chemotherapy for the
treatment of malignancies including multiple myeloma.5 Several investigators have reported that high numbers of NK and LAK
cells are present in PBSC grafts, suggesting a potential role in tumor
eradication after PBSCT.48,49 Although the number of samples is small, we also observed an expansion of the NK
(CD16+) cell population and generation of ADCC activity in
PBSC collections from myeloma patients. Because tumor cells have been
detected in PBSC collections and may contribute to
relapse,50 it might be possible to eliminate myeloma cells
in harvested PBSC by incubation with humanized anti-HM1.24 MoAb.
Furthermore, the combination of current PBSCT and immunotherapy could
well result in enhanced tumor cytotoxicity and improve the prognosis of
multiple myeloma.
In addition to the cytolytic activity of effector cells, there are
certain other advantages to immunotherapy with humanized anti-HM1.24
MoAb in multiple myeloma. First, myeloma cells mainly localize in the
bone marrow, where tumor cells are readily accessible to therapeutic
MoAb.25 Second, there is no soluble HM1.24 antigen in the
serum to block the binding of humanized anti-HM1.24 MoAb. Third,
humanized anti-HM1.24 MoAb does not induce human complement activation,
which could cause serious side effects in patients. Finally, the
ability to generate antibodies against the therapeutic MoAb is likely
to be suppressed because of compromised B-cell function in myeloma
patients.51 These factors suggest that together, immunotherapy using humanized anti-HM1.24 MoAb may be beneficial in
myeloma patients when significant tumor reduction has been achieved by
conventional and/or high-dose chemotherapy. Moreover, our previous
study has shown that anti-HM1.24 MoAb inhibits Ig secretion by human
myeloma cells in SCID mouse models.25 Therefore, humanized
anti-HM1.24 MoAb may also be useful to regulate humoral immunity in a
variety of clinical situations.
In conclusion, the present study demonstrates that humanized
anti-HM1.24 MoAb can mediate myeloma cell-specific cytolysis together
with various effector cells from myeloma patients by an ADCC mechanism,
and that the reduced ADCC activity of effector cells in certain myeloma
patients can be restored by treatment with cytokines, such as IL-2,
IL-12, or IL-15. These results encourage clinical trials with this
humanized MoAb and warrant further investigation into the feasibility
of ex vivo enhancement of ADCC activity by cytokines, especially in
patients with cellular immunodeficiency.
| |
ACKNOWLEDGMENT |
We thank Dr Kevin Boru for review of the manuscript, Drs Shingo
Wakatsuki, Toshiaki Takeichi, Yoshiyuki Miyamoto, Takashi Mizuguchi,
and Yoshihito Okamura for their cooperation, and Tomoko Sei for
excellent technical assistance.
| |
FOOTNOTES |
Submitted June 8, 1998; accepted January 19, 1999.
Supported in part by a grant for Cancer-Induced Bone Diseases from the
Ministry of Health and Welfare, Tokyo, Japan.
The publication costs of this
article were defrayed in part by
page charge payment. This article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. section
1734 solely to indicate this fact.
Address reprint requests to Masaaki Kosaka, MD, PhD, First Department
of Internal Medicine, School of Medicine, University of Tokushima,
3-18-15 Kuramoto-cho, Tokushima 770-8503, Japan.
| |
REFERENCES |
1.
Alexanian R, Dimopoulos M:
The treatment of multiple myeloma.
N Engl J Med
330:484, 1994[Free Full Text]
2.
Bataille R, Harousseau JL:
Multiple myeloma.
N Engl J Med
336:1657, 1997[Free Full Text]
3.
Gahrton G, Tura S, Ljungman P, Blade J, Brandt L, Cavo M, Facon T, Gratwohl A, Hagenbeek A, Jacobs P, de Laurenzi A, Lint MV, Michallet M, Nikoskelainen J, Reiffers J, Samson D, Verdonck L, de Witte T, Volin L:
Prognostic factors in allogeneic bone marrow transplantation for multiple myeloma.
J Clin Oncol
13:1312, 1995[Abstract]
4.
Attal M, Harousseau JL, Stoppa AM, Sotto JJ, Fuzibet JG, Rossi JF, Casassus P, Maisonneuve H, Facon T, Ifrah N, Payen C, Bataille R:
A prospective, randomized trial of autologous bone marrow transplantation and chemotherapy in multiple myeloma.
N Engl J Med
335:91, 1996[Abstract/Free Full Text]
5.
Vesole DH, Tricot G, Jagannath S, Desikan KR, Siegel D, Bracy D, Miller L, Cheson B, Crowley J, Barlogie B:
Autotransplants in multiple myeloma: What have we learned?
Blood
88:838, 1996[Abstract/Free Full Text]
6.
Grossbard ML, Press OW, Appelbaum FR, Bernstein ID, Nadler LM:
Monoclonal antibody-based therapies of leukemia and lymphoma.
Blood
80:863, 1992[Free Full Text]
7.
Vitetta ES, Uhr JW:
Monoclonal antibodies as agonists: An expanded role for their use in cancer therapy.
Cancer Res
54:5301, 1994[Free Full Text]
8.
Ortaldo JR, Woodhouse C, Morgan AC, Herberman RB, Cheresh DA, Reisfeld R:
Analysis of effector cells in human antibody-dependent cellular cytotoxicity with murine monoclonal antibodies.
J Immunol
138:3566, 1987[Abstract]
9.
Brittenden J, Heys SD, Ross J, Eremin O:
Natural killer cells and cancer.
Cancer
77:1226, 1996[Medline]
[Order article via Infotrieve]
10.
Honsik CJ, Jung G, Reisfeld RA:
Lymphokine-activated killer cells targeted by monoclonal antibodies to the disialogangliosides GD2 and GD3 specifically lyse human tumor cells of neuroectodermal origin.
Proc Natl Acad Sci USA
83:7893, 1986[Abstract/Free Full Text]
11.
Saito Y, Iishi Y, Kosaka M:
Analysis of the subsets of Leu-7+ cells in myeloma and their effects on NK cell activity and Ig synthesis by B cells.
Jpn J Clin Immunol
9:441, 1986
12.
Uchida A, Yagita M, Sugiyama H, Hoshino T, Moore M:
Strong natural killer (NK) cell activity in bone marrow of myeloma patients: Accelerated maturation of bone marrow NK cells and their interaction with other bone marrow cells.
Int J Cancer
34:375, 1984[Medline]
[Order article via Infotrieve]
13.
Osterborg A, Nilsson B, Bjorkholm M, Holm G, Mellstedt H:
Natural killer cell activity in monoclonal gammopathies: Relation to disease activity.
Eur J Haematol
45:153, 1990[Medline]
[Order article via Infotrieve]
14.
Gonzalez M, San Miguel JF, Gascon A, Moro MJ, Hernandez JM, Ortega F, Jimenez R, Guerras L, Romero M, Casanova F, Sanz MA, Portero JA, Orfao A:
Increased expression of natural-killer-associated and activation antigens in multiple myeloma.
Am J Hematol
39:84, 1992[Medline]
[Order article via Infotrieve]
15.
Anderson KC, Park EK, Bates MP, Leonard RCF, Hardy R, Schlossman SF, Nadler LM:
Antigens on human plasma cells identified by monoclonal antibodies.
J Immunol
130:1132, 1983[Abstract]
16.
Anderson KC, Bates MP, Slaughengoupt B, Schlossman SF, Nadler LM:
A monoclonal antibody with reactivity restricted to normal and neoplastic plasma cells.
J Immunol
132:3172, 1984[Abstract]
17.
Tong AW, Lee JC, Stone MJ:
Characterization of a monoclonal antibody having selective reactivity with normal and neoplastic plasma cells.
Blood
69:238, 1987[Abstract/Free Full Text]
18.
Stevenson FK, Bell AJ, Cusack R, Hamblin TJ, Slade CJ, Spellerberg MB, Stevenson GT:
Preliminary studies for an immunotherapeutic approach to the treatment of human myeloma using chimeric anti-CD38 antibody.
Blood
77:1071, 1991[Abstract/Free Full Text]
19.
Goldmacher VS, Bourret LA, Levine BA, Rasmussen RA, Pourshadi M, Lambert JM, Anderson KC:
Anti-CD38-blocked ricin: An immunotoxin for the treatment of multiple myeloma.
Blood
84:3017, 1994[Abstract/Free Full Text]
20.
Ellis JH, Barber KA, Tutt A, Hale C, Lewis AP, Glennie MJ, Stevenson GT, Crowe JS:
Engineered anti-CD38 monoclonal antibodies for immunotherapy of multiple myeloma.
J Immunol
155:925, 1995[Abstract]
21.
Goto T, Kennel SJ, Abe M, Takishita M, Kosaka M, Solomon A, Saito S:
A novel membrane antigen selectively expressed on terminally differentiated human B cells.
Blood
84:1922, 1994[Abstract/Free Full Text]
22. Ohtomo T, Sugamata Y, Ozaki Y, Ono K, Yoshimura Y, Kawai S,
Koishihara Y, Ozaki S, Kosaka M, Hirano T, Tsuchiya M: Molecular
cloning and characterization of a surface antigen preferentially
overexpressed on multiple myeloma cells. Biochem Biophys Res Commun (in
press)
23.
Ozaki K, Ozaki S, Kosaka M, Saito S:
Localization and imaging of human plasmacytoma xenografts in severe combined immunodeficiency mice by a new murine monoclonal antibody, anti-HM1.24.
Tokushima J Exp Med
43:7, 1996[Medline]
[Order article via Infotrieve]
24.
Ozaki S, Kosaka M, Harada M, Nishitani H, Odomi M, Matsumoto T:
Radioimmunodetection of human myeloma xenografts with a monoclonal antibody directed against a plasma cell specific antigen, HM1.24.
Cancer
82:2184, 1998[Medline]
[Order article via Infotrieve]
25.
Ozaki S, Kosaka M, Wakatsuki S, Abe M, Koishihara Y, Matsumoto T:
Immunotherapy of multiple myeloma with a monoclonal antibody directed against a plasma cell-specific antigen, HM1.24.
Blood
90:3179, 1997[Abstract/Free Full Text]
26.
Durie BGM, Salmon SE:
A clinical staging system for multiple myeloma; correlation of measured myeloma cell mass with presenting clinical features, response to treatment, and survival.
Cancer
36:842, 1975[Medline]
[Order article via Infotrieve]
27.
Gore ME, Viner C, Meldrum M, Bell J, Milan S, Zuiable A, Slevin M, Selby PJ, Clark PI, Millar B, Maitland JA, Judson IR, Tillyer C, Malpas JS, McElwain TJ:
Intensive treatment of multiple myeloma and criteria for complete remission.
Lancet
2:879, 1989[Medline]
[Order article via Infotrieve]
28. Ono K, Ohtomo T, Yoshida K, Yoshimura Y, Kawai S, Koishihara Y,
Ozaki S, Kosaka M, Tsuchiya M: The humanized anti-HM1.24 antibody
effectively kills multiple myeloma cells by human effector
cell-mediated cytotoxicity. Mol Immunol (in press)
29.
Sato K, Tsuchiya M, Saldanha J, Koishihara Y, Ohsugi Y, Kishimoto T, Bendig M:
Humanization of a mouse anti-human interleukin-6 receptor antibody comparing two methods for selecting human framework regions.
Mol Immunol
31:371, 1994[Medline]
[Order article via Infotrieve]
30.
Ohtomo T, Tsuchiya M, Sato K, Shimizu K, Moriuchi S, Miyao Y, Akimoto T, Akamatsu K, Hayakawa T, Ohsugi Y:
Humanization of mouse ONS-M21 antibody with the aid of hybrid variable regions.
Mol Immunol
32:407, 1995[Medline]
[Order article via Infotrieve]
31.
Mizuguchi T, Kosaka M, Saito S:
Activin A suppresses proliferation of interleukin-3-responsive granulocyte-macrophage colony-forming progenitors and stimulates proliferation and differentiation of interleukin-3-responsive erythroid burst-forming progenitors in the peripheral blood.
Blood
81:2891, 1993[Abstract/Free Full Text]
32.
Velde AA, Malefijt RW, Huijbens RJF, de Vries JE, Figdor CG:
IL-10 stimulates monocyte FcR surface expression and cytotoxic activity: Distinct regulation of antibody-dependent cellular cytotoxicity by IFN-, IL-4, and IL-10.
J Immunol
149:4048, 1992[Abstract]
33.
Baldwin GC, Chung GY, Kaslander C, Esmail T, Reisfeld RA, Golde DW:
Colony-stimulating factor enhancement of myeloid effector cell cytotoxicity towards neuroectodermal tumour cells.
Br J Haematol
83:545, 1993[Medline]
[Order article via Infotrieve]
34.
Massaia M, Bianchi A, Dianzani U, Camponi A, Attisano C, Boccadoro M, Pileri A:
Defective interleukin-2 induction of lymphokine-activated killer (LAK) activity in peripheral blood T lymphocytes of patients with monoclonal gammopathies.
Clin Exp Immunol
79:100, 1990[Medline]
[Order article via Infotrieve]
35.
Gottlieb DJ, Prentice GR, Mehta AB, Galazka AR, Heslop HE, Hoffbrand AV, Brenner MK:
Malignant plasma cells are sensitive to LAK cell lysis; Pre-clinical and clinical studies of interleukin 2 in the treatment of multiple myeloma.
Br J Haematol
75:499, 1990[Medline]
[Order article via Infotrieve]
36.
Vooijs WC, Schuurman H-J, Bast EJEG, de Gast GC:
Evaluation of CD38 as target for immunotherapy in multiple myeloma.
Blood
85:2282, 1995[Free Full Text]
37.
Riechmann L, Clark M, Waldmann H, Winter G:
Reshaping human antibodies for therapy.
Nature
332:323, 1988[Medline]
[Order article via Infotrieve]
38.
van de Winkel JGJ, Capel PJA:
Human IgG Fc receptor heterogeneity; Molecular aspects and clinical implications.
Immunol Today
14:215, 1993[Medline]
[Order article via Infotrieve]
39.
Eisenthal A, Rosenberg SA:
Systemic induction of cells mediating antibody-dependent cellular cytotoxicity following administration of interleukin 2.
Cancer Res
49:6953, 1989[Abstract/Free Full Text]
40.
Soiffer RJ, Robertson MJ, Murray C, Cochran K, Ritz J:
Interleukin-12 augments cytolytic activity of peripheral blood lymphocytes from patients with hematologic and solid malignancies.
Blood
82:2790, 1993[Abstract/Free Full Text]
41.
Carson WE, Giri JG, Lindemann MJ, Linett ML, Ahdieh M, Paxton R, Anderson D, Eisenmann J, Grabstein K, Caligiuri MA:
Interleukin (IL) 15 is a novel cytokine that activates human natural killer cells via components of the IL-2 receptor.
J Exp Med
180:1395, 1994[Abstract/Free Full Text]
42.
Baxevanis CN, Tsavaris NB, Papadhimitriou SI, Zarkadis IK, Papadopoulos NG, Bastounis EA, Papamichail M:
Granulocyte-macrophage colony-stimulating factor improves immunological parameters in patients with refractory solid tumours receiving second-line chemotherapy; Correlation with clinical responses.
Eur J Cancer
33:1202, 1997
43.
Bataille R, Jourdan M, Zhang XG, Klein B:
Serum levels of interleukin 6, a potent myeloma cell growth factor, as a reflect of disease severity in plasma cell dyscrasias.
J Clin Invest
84:2008, 1989
44.
Reibnegger G, Krainer M, Herold M, Ludwig H, Wachter H, Huber H:
Predictive value of interleukin-6 and neopterin in patients with multiple myeloma.
Cancer Res
51:6250, 1991[Abstract/Free Full Text]
45.
Urashima M, Ogata A, Chauhan D, Hatziyanni M, Vidriales MB, Dedera DA, Schlossman RL, Anderson KC:
Transforming growth factor-1; differential effects on multiple myeloma versus normal B cells.
Blood
87:1928, 1996[Abstract/Free Full Text]
46.
Yamamoto N, Zou JP, Li XF, Takenaka H, Noda S, Fujii T, Ono S, Kobayashi Y, Mukaida N, Matsushima K, Fujiwara H, Hamaoka T:
Regulatory mechanisms for production of IFN- and TNF by antitumor T cells or macrophages in the tumor-bearing state.
J Immunol
154:2281, 1995[Abstract]
47.
Bellone G, Aste-Amezaga M, Trinchieri G, Rodeck U:
Regulation of NK cell functions by TGF-1.
J Immunol
155:1066, 1995[Abstract]
48.
Neubauer MA, Benyunes MC, Thompson JA, Bensinger WI, Lindgren CG, Buckner CD, Fefer A:
Lymphokine-activated killer (LAK) precursor cell activity is present in infused peripheral blood stem cells and in the blood after autologous peripheral blood stem cell transplantation.
Bone Marrow Transplant
13:311, 1994[Medline]
[Order article via Infotrieve]
49.
Silva MRG, Parreira A, Ascensao JL:
Natural killer cell numbers and activity in mobilized peripheral blood stem cell grafts: Conditions for in vitro expansion.
Exp Hematol
23:1676, 1995[Medline]
[Order article via Infotrieve]
50.
Lemoli RM, Fortuna A, Motta MR, Rizzi S, Giudice V, Nannetti A, Martinelli G, Cavo M, Amabile M, Mangianti S, Fogli M, Conte R, Tura S:
Concomitant mobilization of plasma cells and hematopoietic progenitors into peripheral blood of multiple myeloma patients; Positive selection and transplantation of enriched CD34+ cells to remove circulating tumor cells.
Blood
87:1625, 1996[Abstract/Free Full Text]
51.
Pilarsky LM, Andrews EJ, Mant MJ, Ruether BA:
Humoral immune deficiency in multiple myeloma patients due to compromised B-cell function.
J Clin Immunol
6:491, 1986[Medline]
[Order article via Infotrieve]
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ABSTRACT
INTRODUCTION