Microbiol. Immunol. Vol. 36 (12), 1305-1316, 1992
Establishment of Stable Cell Lines Producing AntiPseudomonas aeruginosa Monoclonal Antibodies and Their Protective Effects for the Infection in Mice Hisayoshi O'OKA,*,1,4 Eiko CHONAN,1 Kazunori MIZUTANI,1,5 Tamotsu FUKUDA,2 Yasuyuki KUROIWA,2 Yasushi ONO,3 and Shiro
SHIGETA1
1Department of Microbiology,Fukushima Medical College,Fukushima 960-12, Japan,2Life Science
Laboratory, Mitsui Toatsu Chimical Inc., Mobara, Chiba 297, Japan, 3Departmentof Microbiology,Nihon UniversitySchoolof Medicine, Itabashi-ku, Tokyo 173, Japan, 4Institute of Biological Science,Mitsui Pharmaceutical Inc., Mobara, Chiba 297, Japan, and 5Departmentof Orthopedics, Tohoku UniversitySchoolof Medicine, Sendai, Miyagi 980, Japan (Accepted for publication, September 29, 1992)
Abstract
Human-human
specific P.
for
five
aeruginosa
P109
primed
cells
(10
ranged
0.5
commercial
bacterial in the
effects
the ratio
IgG
of
per tion of
cells
of
anti-P.
patients
in
hr.
P.
P.
the
more It
and
times
the
months to
such
use MoAbs
of
P.
and these
their
must
10
lines be
useful
for
to
for
as to
60 ƒÊg
large-scale the
in showed
well
capacity
produced cell
protective decreased
MoAbs as
into
a macrophage
were
aeruginosa
maintained
than promoted
showed
All
P.
MoAbs
invasion
which (CY).
of
mice
by
also
mice
the of
protective
trapped
MoAbs
in
isolates
12
more
bacterial
bacteria
bacteria.
lines
values
to
prevented of
by
challenge
(ED50)
240
fusing
myeloma
produced
(i.p.)
cyclophosphamide
is practicable
MoAbs
aeruginosa
and
cell
than
to
by
human
administration
number
clinical
with were
dose
26
aeruginosa
by
hybridoma
for 24
aeruginosa with
to
The
continuously 106
the trapped
of
(PMN)
binding strains.
were
cavity, both that
cells
effective
MoAb
peritoneal
infection
which
(MoAbs)
developed
intraperitoneal
50% and
antibodies were
cells
MoAbs
preparation.
macrophages
lethal
serotype-specific
MoAb
The
of increasing
polymorphonuclear
immunized
mice.
in
way
against
The lethal
10.2 ƒÊg/mouse
clearance
blood and
to
human
aeruginosa
virus-transformed
against
in
monoclonal
Pseudomonas
Epstein-Barr
protective
LD50)
from
producing
of
polyethyleneglycol.
were
aeruginosa
serotypes and
using
hybridomas
a
hybridomas
major
to
the
produce MoAbs produc-
therapeutics
infection.
Pseudomonasaeruginosainfection in patients with major thermal injury (12) or with leukemia (21) have resulted in high mortality despite intensive antibiotic therapy. To such cases immunological therapy for P. aeruginosa infections should be of great medical benefit (17). Passive immunization for P. aeruginosa has been reported to be effective for prophylaxis and cure of experimental infection of P. aeruginosa (4, 13). For clinical use, plasma-derived human immunoglubulins with 130 5
130 6
H.
O'OKA
ET AL
naturally high titers of antibodies to P. aeruginosahave been examined but their efficiencies are variable by serotype or mucoidization of strain, and moreover they are limited in supply (20, 27). As an alternative of plasma-derived polyclonal antibodies, several human monoclonal antibodies (MoAbs) to P. aeruginosaantigens have been developed (10, 14, 18, 23, 24, 28) and they are expected to be a plentiful source of potential antibodies. Since most of these MoAbs producing cells are derived from hybridomas between sensitized human lymphocytes and murine origin myeloma cells, one has to be cautious for the contamination of murine cell products in the MoAbs. Some of the MoAbs have been derivedfrom Epstein-Barr virus (EBV)transformed human lymphoblastoid cell lines (LCL); however, these EBV-transformed LCLs are ordinarily unstable in either or both of cell proliferation and antibody production. We have developed human MoAbs against major serotypes o P.aeruginosa using EBV-transformed LCL of human lymphocytes sensitized toP.aeruginosa. To enable large amount of MoAb production, several hybridoma cell lines were established by fusion of the EBV-transformed cells and established human myeloma P109 cells. In this study, we report a successful establishment of the stable cell lines producing human MoAbs against five Homma serotype strains of P. aeruginosa,i.e., A, B, E, G and I, which cover more than 70% of clinical isolates ofP.aeruginosa in Japan (15). We have evaluated therapeutic effect of these MoAbs against P. aeruginosa septicemia in mice and their specificity to several clinical isolates. MATERIALS
AND
METHODS
P. aeruginosa. Homma serotype standard strains (ATCC 27577-27590) were purchased from American Type Culture Collection (Bethesda, Md., U.S.A.). PA103 strain, IID 5004 and IID 5018 were provided kindly by Dr. J.Y. Homma, Kitasato Institute, Tokyo. The other strains were isolated in Fukushima medical college hospital. All strains were cultured in heart infusion agar (Nissui Pharmaceuticals, Tokyo) or Homma's synthetic medium (7). Serotypings were done using murine MoAbs for P. aeruginosa,"Mei-Assay" (Meiji Seika Co., Ltd., Tokyo), according to the instruction by the Serotyping Committee ofJapanPseudomonas aeruginosasociety (7). Mice. Female BALB/cCrSlc mice, 8 to 10 weeks old were purchased from Japan SLC (Hamamatsu, Japan) and were used throughout the study. Antibodypreparation. Procedures for preparation of serotype-specific human MoAbs against five major serotypes of P. aeruginosa were as follows. First we established LCLs which produced MoAbs against each Homma's serotype-strains with immortalized peripheral blood lymphocytes from healthy volunteers by EBV infection. After the establishment of transformed cells, the MoAb-producing cells were fused with human myeloma cells (P109, HGPRT-, ouabain resistant) using polyethyleneglycol (m.w. 4,000) (1). After several cycles of clonings of MoAbproducing cells, cultures were scaled up to 50 liters of cell suspensions. For the
HUMAN
MONOCLONAL
ANTIBODY
FOR
P. AERUGINOSA
130 7
production of MoAb from the hybridoma cells a serum-free NYSF-404 medium (26) was used. The supernatants of each hybridoma cell culture were pooled, precipitated with ammonium sulfate (final saturated 50%) and dialysis against phosphate-buffered saline (PBS). The isotype of each MoAb was determined by dot-immunobinding assay with rabbit antibodies to hnman IgA, IgG and IgM. All MoAbs produced were identified as IgM isotype. The quantity of IgM was determined with ELISA. Dot-immunobindingassay (DIBA). Binding of MoAbs to P. aeruginosacells was determined with DIBA (5). In brief, formalin-killed bacterial cell suspension was dotted on nitrocellulose membrane filter. Then the cells were reacted first with MoAb and second with a peroxidase-conjugated anti-human immunoglobulin rabbit serum (Dakopatt, Glostrup, Denmark) each for 30 min at 37 C. We examined the peroxidase activity with 4-chloro-l-naphtol and hydrogen peroxide and the positive reaction was detected as brown-colored dot. Agglutination test. The live-cell slide-agglutination according to Homma (6) was employed. Bacterial cells grown on heart-infusion agar plate were picked up with a toothpick and mixed with a drop of each MoAb for major serotypes of P. aeruginosa on a slide glass. When definite agglutination was observed within 60 sec, it was judged to be positive. Protectiveactivity against P. aeruginosa infection. Groups of 5 to 10 mice were injected i.p. with 0.5 ml of serially diluted human MoAb. Two hours after injection the animals were challenged with 0.5 ml of approximately 10 LD50 of P. aeruginosa i.p. Colony-forming units of P. aeruginosawhich expressed 1 LD50 were as follows : type A-P5, 2 x 105; type B-P12, 1 x 104; type E-PA103, 5 x 104; type G-FP8, 7 x 104; type I-P35, 5 x 104. The animals were observed for 6 days after challenge. The mean 50% effective dose (ED50), i.e., the minimal dose of a MoAb needed to protect the death by infection in half of the animals of a group, was calculated by LitchfieldWilcoxon method (11). As a model of immunodeficient hosts, mice were injected i.p. with a single dose of 200 mg/kg cyclophosphamide (CY, Shionogi Pharmaceutical Ltd.). At 4 days after injection of CY, the average number of PMN in peripheral blood decreased to 1/10 of that at start of CY treatment. The mice were treated with MoAbs and challenged with P. aeruginosaat 4 days after CY treatment following the procedures described above. Details of the effect of CY on peripheral leukocytes of mice were reported previously (8). In vivo phagocytictest. Mice were pretreated by i.p. injection of 0.5 ml of 5% thioglycolate for induction of phagocytic cells. Three days later, mice were injected i.p. with equal 0.5 ml volume mixture of human MoAb N1-3 (anti-type E and F, 1 or 10 ,ug/m1) and P. aeruginosa PA103 (serotype E) suspension (109 cfu/ml). As control, equal volume mixture of PBS and bacterial suspension was used. All mixtures were injected to mice immediately after the preparation. At 30 min after injection, peritoneal exudate was withdrawn and peritoneal cavity was washed repeatedly with PBS. The exudate and all washing fluid were combined and cells were collected by low speed centrifugation, smeared on slide glass, Giemsa-
130 8
H.
O'OKA
ET AL
stained, and were examined microscopically for morphology and phagocytosis of bacteria. Enumeration of bacteria in blood stream and peritoneal cavity. Mice were injected with 5 ,ug of MoAb (N1-3, anti-type E and F) or PBS into peritoneal cavity and 2 hr later 5 x 105 cfu of P. aeruginosaPA103 (serotype E) were injected at the same place. Washings from peritoneal cavities in 3 ml of PBS and heart blood samples were examined for the number of viable P. aeruginosa using heart-infusion agar plates. Colonies of organism were counted after 20 hr of incubation of the plates at 37 C. RESULTS Productionof Human MoAbs We established 26 human LCLs which produced MoAbs to P. aeruginosa 0antigens from 80 preparations of EBV-infected human peripheral lymphocytes. These MoAbs were examined for in vivo protective activity against P. aeruginosa infection in mice using respective type-specific strain. As a result we selected 6 LCLs which produced the most effective MoAbs against infection of each serotype i.e., No. 13 against serotype A, No. 12 against serotype B, No. 5 and 9 against serotype E, No. 21 against serotype G, and No. 2 against serotype I. These LCLs were fused with P109 cells as described in "MATERIALS AND METHODS." Two to 3 weeks after the fusion of cells, the hybridomas started to grow and had been producing serotype-specific human MoAbs for more than 12 months continuously. After several cycles of clonings of the hybridoma cell lines, 6 largescale cell cultures were established. The MoAbs obtained from the supernatant of the cultures were designated as V4-7 to type A and F, V3-4 to type B, N1-3 to type E and F, V9-1 to type E, N8-6 to type G and V5-1 to type D and I (Table 3). Stability of AntibodyProductionof HybridomaCell Lines The MoAb-producing hybridoma cell lines were frozen and stored in liquid nitrogen container after the scale-up of cultures. We examined MoAb-producing capacity of the cells in serum-free medium at the recovery of cells, at 3 and 12 months of culture after the recovery. These established human hybridomas grew Table
1.
Stability
of MoAb-producing
hybridomas
HUMAN
MONOCLONAL
Table
2.
ANTIBODY
Prophylactic
and
therapeutic
P. aeruginosa
well
in
serum-free
MoAbs fied
for
medium
more
from
the
than
and
1 year
supernatant
of
the
of MoAb
producing At
cultures
130 9
against
to mice
been 1).
P. AERUGINOSA
effect
infection
had
(Table
FOR
constant
3 months
and
amount
of culture,
were
of
MoAbs
examined
for
human
were
anti-P.
puri-
aeruginosa
activities.
Prophylactic
and
Therapeutic
Eight-week-old N1-3
at various
strain, was P.
times
serotype
signs.
When
or
and
we
before
or
infection.
were
prevented
day
not
compared
EC50
of
from with
MoAbs
and
2
hr
against
aeruginosa
i.p.
ment
died
within
infections
were
ED50
of
pooled Thus
human MoAbs
although
Lethal
Challenges
challenged
by
injection
(Table
2
days.
The
3). ED50
0.5-1.9ƒÊg/mouse
10.2 ƒÊg/mouse. serum were
was 26
the
more to 240
than times
time
mice.
When
were
prevented
and
showed
no
infection,
the
mice
death
was
MoAb from
manifested were
of the mice was 4 hr after infection,
to
MoAb (PA103
bad
delayed
also on
the
the mice
about
one
2).
by P.
aeruginosa
were
injected
10
LD50
of
infected
values
other
of
human
aeruginosa
mice
the
after
Mice
5 pg
of P.
mortality
hr
in
with
LD50
condition was given
All
except On
2
the
were
11.2
condition
(Table
mice
i.p.
good at
group
i.p.
infection
general MoAb
BALB/c
they
with
in
the When
death,
female
later
were
non-treated
Eight-week-old
of
administered
from death although following infection.
with for
time
Infection
injected
examined
the
mice
protected next day
aeruginosa were
challenge
daily
was
P.
mice
after
at
All
MoAb
against
BALB/c
before
E)
administered aeruginosa
Effect
female
of
mice
these
anti-type hand
i.p.
64 ƒÊg/mouse more effective
human
ED50 against than
IgG
P.
treat-
type-specific
V3-4,
of
of
antibody
against
MoAb
MoAbs,
serotype
without
MoAbs B
the
with
corresponding
which
showed
preparation
all serotypes' polyclonal
from
infections. human IgG
preparation. Immunodeficient amined after
for CY
the
treatment.
mice protective
which effects
LD50s
of
were of
P.
MoAbs
aeruginosa
injected
with
against type
P. E
200
mg/kg
aeruginosa
(PA103)
and
of
CY
infection type
I (P35)
were at
ex-
4 days against
131 0
H.
Table
3.
Protective
Table
4.
activity
of MoAbs
Protective
mice
administration mice
and
EC50
of
of all
mice
MoAb
parable
to
and
type
MoAbs
to
clarify
to
mice,
we
blood F) E).
for
order
infection
E
of
of mice to
10 In
the
for the
after
group
V5-1 and
infection
infection.
of
2 hr mice
P.
died I,
before
D)
was
within
against
in
of
effect bacteria
infection
antibody
after
3
they
days
of
by almost
mice
(Tables
MoAbs
to
the
infection.
infection were
the to
their com-
3,
4).
vivo
administered the
hours challenged
the
and
immunocompetent
clearance
without
Two
aeruginosa
MoAb
of protective
We
P. aeruginosa
respectively.
(type
by P. aeruginosa
mice
of
Macrophages
the
against
1.34 ƒÊg/mouse in
mechanism
mouse
cfu
each with
and
Mice
of MoAbs
2.5
LD50
0.46
examined
of each the
E)
were
EC50
Activity In
heart
the
and
4
treatment
(type
serotypes
i.p. lethal infection
in immunodeficient
1.5
i.p.,
without
V9-1
counterparts
Opsonic
were
MoAbs
ET AL
against
activities
infection
immunodeficient
O'OKA
of from
PBS of P.
or
5ƒÊg
aeruginosa
administration,
P.
peritoneal
aeruginosa cavity
of N1-3 (PA103 viable
and
(anti-type strain, bacteria
HUMAN
MONOCLONAL
ANTIBODY
(a ) peritoneal cavity
Fig.
1.
Clearance
of MoAb two
to
hours
cavities of
for
of organism
injected shaded
were
represents PBS.
105
Each
the
cfu 3 ml
of P.
with
of
counted
after
and from
viable 20
cavity 5 tig
aeruginosa
of PBS
number
undetectable
peritoneal
injected
sample
mean•}S.E. The
from
were
with
infection.
examined
bar
5x
P. AERUGINOSA
131 1
(b )healrt blood
aeruginosa
Mice
before
peritoneal hours
of P.
mice.
FOR
hr
(N=3). or
PA103
heart both
P.
and
aeruginosa of incubation
samples
, mice
uncountable
level
E)
culture injected of
bacteria
at
and
or
PBS
37
C.
Each
represented
at
from indicated
blood
plates.
MoAb; •›, is
the
heart
agar at
F)
Washings
obtained
heart-infusion
administration E,
infection.
cavities
of the
Symbols: •œ
after
anti-type
were
peritoneal using
blood
(N1-3,
(serotype
blood the
heart
of MoAb
was
Colonies point
and
control
mice by
the
area.
in peritoneal cavity had gradually decreased to 1/10 of start at 6 hr after infection. However, at 24 hr after infection, the number of viable bacteria reached to more than 106 cfu/ml. In heart blood, 103 viable bacteria appeared at 30 min after infection, gradually increased up to 6 hr and reached to more than 106 cfu/ml at 24 hr. Those mice showed septic state at 24 hr after infection and died. On the other hand, in the group of MoAb-treated mice, the number of viable bacteria in peritoneal cavity decreased to 1/3 at 30 min and to 1/200 at 6 hr after infection. After 24 hr, no viable bacteria were detected in peritoneal cavity. In heart blood
131 2
H.
O'OKA
ET AL
Fig. 2. Phagocytosis of P. aeruginosa by peritoneal macrophages in infected mice. Mice were injected with 0.5 ml of 5% thioglycolate i.p. for the induction of phagocytic cells. At 3 days after injection, 1 ml of equal mixture of P. aeruginosa PA103 (serotype E) suspension (109 cfu/ml) and human MoAb N1-3 (anti-type E, F, 1 or 10 tig/m1) or PBS (control) was introduced i.p. At 30 min after challenge, peritoneal exudate was withdrawn by washing with PBS, smeared on slide glass, Giemsa-stained, and examined by lightmicroscopy for the phagocytosis of bacteria by macrophoges. More than 100 macrophages were examined.
of the treated mice, viable bacteria were less than 100 cfu/ml after 1 hr of infection (Fig. 1). When we examined peritoneal cells in Giemsa-stained preparation at 30 min after i.p. injection of the mixture of MoAbs (or PBS) and bacteria, most of PMN were broken probably by their fragile nature after phagocytosis and more than 90% of the intact cells were macrophages. In the control group of mice injected with PBS and bacteria, only 3% of macrophages trapped bacterial particles and the average number of bacteria in phagocytozing cells was 4. On the other hand, in the group injected with MoAb and bacteria, the rate of macrophages phagocytozed more than one bacterial particle was over 80%. In addition, the mode of the bacterial number trapped in macrophages increased to 5 to 6 according to the antibody dose increment (Fig. 2). Thus antibody administration to mice promoted active phagocytosis of intraperitoneal macrophages, that is, in the numbers of bacteria trapped by a macrophage and in the ratio of macrophages trapped bacteria. Specificityof MoAbs to Clinical Strains of P. aeruginosa We examined randomly selected 54 clinical isolates for the reactivity to the MoAbs prepared. Specificities of the MoAbs to respective serotype were examined by agglutination test. The serotypes of the clinical isolates were previously deter-
HUMAN
Fig.
3. No
Slide
agglutination
agglutination
Agglutination ・,
MONOCLONAL
agglutination was
was was
of
clinical
observed
observed
to
ANTIBODY
to both
isolates all anti-G
of P.
antibodies and
FOR
aeruginosa in
M.
P. AERUGINOSA
with
a serotyping
Symbols; •œ
MoAbs. kit.
131 3
a Non-typable 1) Polyagglutinable
, agglutination
was
: :
positive,
negative,
mined by Mei-Assay. MoAbs N1-3 and V5-1 were agglutinable all the tested clinical isolates corresponding to the respective serotype. MoAb V9-1 was agglutinable all serotype E isolates and one of four serotype F strains. MoAb N8-6 was agglutinable all serotype G isolates and one poly-agglutinable (type G and M) strain. MoAb V3-4 was agglutinable five of six type B strains and one non-typable strain. Thus all 54 clinical isolates except of 1 strain of type B and 3 strains of type M were reactive with either one of the 6 MoAbs used (Fig. 3). DISCUSSION We developed stable cell lines producing human MoAbs against P. aeruginosa. There have been two different methods to establish cell lines which produce human MoAbs. One is fusion method (2, 16), and the other is EBV transformation method (22, 25). Although the fusion method is well established for producing murine MoAbs, direct fusion of human MoAb-producing lymphocytes and cultured myeloma cells is rather difficult because hyperimmunization for P. aeruginosa does not occur during the course of natural infection. Further, human origin myeloma cell lines had lower fusion efficiency than that of murine origin (26). Transformation method with EBV infection gives highly efficient establishment antibody-producing human LCL. However, antibody production and growth stability of LCL are generally low. Most of our established LCLs were low antibody producer except No. 2, No. 5 and No. 12 (data not shown). Hence we tried the EBV-hybridoma method (1), which combined the transformation method and the fusion method. In this method we could screen MoAbs of LCL for the protective activity against P. aeruginosainfection prior to fusion with myeloma cells. Using this method we could select the most suitable clones for the fusion with myeloma cells. The MoAbs we developed here have been obtained from 3 months cultures of
131 4
H.
hybridoma
cells
However,
3 MoAbs
It
is considered
chain of
and
of
P.
LPS
N1-3,
that
the
recognition
the
report
LPS and
acetylfucosamine.
It
(IATS
11)
which
reacts
vasamin
and
F
with
or
values
of
1
is considered 4)
all
MoAbs
the
polyclonal IgG more effective
preparation was than polyclonal
anti-B
B
serotype
serotypes
LPS
as
reported
opsonins,
and
killing
of P.
mice
against
The
result
in
cavity
The
workers
CY
the
and
structures
4
share
L-N-
and
D-N-
reacts
with
and 9)
serotype
that
recognizes
MoAb
E V5-1
n-N-acetylquino-
serotype-specific
MoAbs
protection model
B).
were On
clear
2,
but
5,
16,
0.5
the
of
MoAbs of low also
less
to ED50
except
ED50
workers was
prior The
1.9 ƒÊg/mouse hand,
Thus the reasons
other
MoAb
mice
death.
to
other
The
the
20)
to
from
human
were much efficacy of
reported
that
than
other
effective
polyclonal the
aeruginosa
is
P.
1
shows
an
The MoAb
opsonic
by
lethal
against
of
phagocytosis
its
phagocytosis
aeruginosa
important
of
and
of
through
which
2 also supported phagocytosis by
specific
antibody
of P.
aeruginosa
polymorphonuclear
also
promote
the
ability
(3).
enhancement
factor Fig.
0-antigen by
(27),
infection
that
result in promoted
effect
antibodies
efficiency
on
bacterial the
clearance MoAbs
this concept macrophages
the
latter
from
exert
their
and confirmed in addition
was
reported
by
to
other
(27). MoAbs
by
the
11
which
(IATS
significant
is not IATS
that
Fig.
protective effects. that administered PMNs.
N1-3
64 to 270 gig/mouse. IgG preparation.
promote
protect
peritoneal
serotype
28).
been
act
cells to
has
3,
0-polysaccharide
determined
epitopes.
(anti-type
V3-4
(Fisher
(19,
It
MoAb
on
together.
D-N-acetylquinovasamin
D
experimental
V3-4
strains.
serotypes
are
they
IATS
MoAb
of
of
MoAbs
which
share
1) and
in
10.4 ƒÊg/mouse
anti-serotype
in
serotype
2 different
L-N-acetylfucosamine
(IATS
resulted
in
them,
that
as
MoAb
to 9
administration i.p.
al
recognizes I
of
et
and
corresponding
with
epitopes
D-N-acetylfucosamine
challenge
reacted
Knirel
IATS
(IATS
with
V5-1)
According
serotype
Intraperitoneal bacterial
of
(9).
a cetylfucosamine,
ET AL
type-specifically
(V4-7,
by
aeruginosa
reacted
O'OKA
protected
the
administration
above-mentioned
elimination
of on
which
phagocytozed
phage.
by
macrophages
promote
The
activation
macrophages
in
that
of
phagocytic
of
complements
peritoneal
P.
and
the by
may cavity.
both
increase
of
of
of
promote
of
in and
through
be
a
P.
C3b
chemotaxis
should
the
effect
of
macrophages
bacteria
antibody
the
possibilities
in
opsonic
ratio
number IgM
scarce supports
participate
increased
of
were
evidence
The
macrophages
also These
PMN
This
MoAbs.
the
complexes
activity
which
macrophages of
by
in
aeruginosa.
peritoneal
administration
complements
the
by
confirmed
bacteria
mice
infection
the
was
Activation
may
lethal
concept bacteria
MoAb
immunocompromised
from
of clarified
macro-
aeruginosa receptors. PMN in
and further
experiments. The
MoAbs
agglutination than protect
agglutinated spectrum
92%
of the
54
clinical
infection
several
of
these
isolates by
these
MoAbs panel. clinical
clinical against So
isolates five
it is considered
isolates.
The
serotype-specifically. major
serotypes that
human
these
Total covered MoAbs
MoAb-producing
more can
also cell
HUMAN
MONOCLONAL
ANTIBODY
FOR
P. AERUGINOSA
131 5
lines which were presented in this report continuously produced MoAbs for more than 12 months and we have been successful in a large-scale culture of cells and a large amount of production of anti-P. aeruginosa MoAb. Our results presented in this study indicate the potential of human MoAb therapy for infections with P. aeruginosa. These hybridoma cell lines that secrete stably high-titered human MoAbs against major serotypes of P. aeruginosa will be useful for clinical therapeutics against P. aeruginosa infections. REFERENCES
1) Campling, B.G., Cole, S.P.C., Atlaw, T., Roder, J.C., and Kozbor, D. 1987. The EBV hybridoma technique and its applications. In Human Hybridomas, Marcel Dekker, Inc., New York. 2) Croce, C.N., Linnenbach, A., Hall, W., Strepwski, Z., and Koprowski, H. 1980. Production of human hybridomas secreting antibodies to measles virus. Nature 288: 488-489. 3) Crytz, S. J., Jr., Furer, E., and Germanier, R. 1983. Protection against Pseudomonasaeruginosa infection in a murine burn wound sepsis model by passive transfer of antitoxin A, antielastase, and antilipopoly-saccharide. Infect. Immun. 39: 1072-1079. 4) Fisher, M.W., and Manning, M.C. 1958. Studies on the immunotherapy of bacterial infections. I. The comparative effectiveness of human-globulins against various bacterial species in mice. J. Immunol. 81: 29-31. 5) Hawkes, R., Niday, E., and Gordon, J. 1982. A dotimmunobinding assay for monoclonal and other antibodies. Anal. Biochem. 119: 142-147. 6) Homma, Y.J. 1976. A new antigenic schema and live-cell slide agglutination procedure for the intrasubspecific, serological classification of Pseudomonasaeruginosa.Jpn.J. Exp. Med. 46: 329-336. 7) Homma, J.Y., Ghoda, A., Goto, S., Kato, I., Kodama, H., Kosakai, N., Kono, M., Shinoya, H., Terada, Y., Tomoyama, T., and Yabuuchi, E. 1979. Proposal of an international standard for the intrasubspecific serological classification of Pseudomonasaeruginosa. Jpn. J. Exp. Med. 49: 89-94. 8) Hyodo, S., Katahira, S., and Shigeta, S. 1982. Experimental infection of immunocompromised mice with Achromobacterxylosoxidans.Tohoku J. Exp. Med. 136: 251-261. 9) Knirel, Y.A., Vinogradov, E.V., Kocharova, N.A., Paramonov, N.A., Kochetkov, N.K., Dmitriev, B.A., Stanislaysky, E.S., and Lanyi, B. 1988. The structure of O-specific polysaccharides and serological classification of Pseudomonasaeruginosa (a review). Acta Microb. Hung. 35: 3-24. 10) Lang, A.B., Furer, E., Larrick, J.W., and Cryz, S. J., Jr. 1989. Isolation and characterization of human monoclonal antibody that recognize epitopes shared by Pseudomonasaeruginosa immunotype 1, 3, 4 and lipopolysaccharides. Infect. Immun. 57: 3851-3855. 11) Litchfield, J.T., Jr., and Wilcoxon, F. 1949. A simplified method of evaluating dose-effect experiments. J. Pharmacol. Exp. Thera. 96: 99-113. 12) McManus, W.F., Goodwin, C.F., Mason, A.O., and Pruitt, B.A. 1981. Burn wound infection. J. Trauma. 21: 753-756. 13) Millican, R.C., Rust, J., and Rothental, S.M. 1957. Gammaglobulin factors protective against infections from Pseudomonasand other organisms. Science 126: 509-511. 14) Ochi, H., Ohtuka, H., Yokota, S., Uezumi, I., Terashima, M., Irie, K , and Noguchi, H. 1991. Inhibitory activity on bacterial mobility and in vivo protective activity of human monoclonal antibodies against flagella of Pseudomonasaeruginosa. Infect. Immun. 59: 550-554. 15) Oguri, T., Inoue, K., and Sato, Y. 1991. Annual changes of serotype distribution and drug sensitivity of Pseudomonasaeruginosa, p. 111-114. In Proceeding of the 24th meeting of the Japan Pseudomonasaeruginosa Saciety, The Japan Pseudomonasaeruginosa Society, Tokyo. 16) Olsson, L., and Kaplan, L. 1980. Human-human hybridomas producing monoclonal antibodies of predefined specificity. Proc. Natl. Acad. Sci. U.S.A. 77: 5429-5431.
131 6
17) 18)
19)
20) 21)
22) 23)
24)
25) 26) 27) 28)
H.
O'OKA
ET AL
Pennington, J.E. 1990. Pseudomonasaeruginosa immunotherapy. Eur. J. Clin. Microbiol. Infect. Dis. 9: 377-380. Sawada, S., Suzuki, M., Kawamura, T., Fujinaga, S., Masuho, Y., and Tomibe, K. 1984. Protection against infection with Pseudomonasaeruginosa by passive transfer of monoclonal antibodies to lipopolysaccharides and outer membrane proteins. J. Infect. Dis. 150: 570 -576. Sawada, S., Kawamura, T., and Masiho, Y. 1987. Immunoprotective human monoclonal antibodies against five major serotypes of Pseudomonasaeruginosa. J. Gen. Microbiol. 133: 3581 3590. Schiller, N.L., Alazard, M. J., and Borwski, R.S. 1984. Serum sensitivity of a Pseudomonasaeruginosa mucoid strain. Infect. Immun. 45: 748-755. Schmpff, S.C., Young, V.M., Greene, W.H., Vermeulen, G.D., Moody, M.R., and Wiernik, P.H. 1972. Origin of infection in acute nonlymphocytic leukemia, significance of hospital acquisition of potential pathogens. Ann. Internal Med. 77: 707-714. Steintz, M., Klein, G., and Koskmies, S. 1977. EB virus-induced B lymphocyte cell lines producing specific antibody. Nature 269: 420-422. Suzuki, H., Okubo, Y., Moriyama, M., Sasaki, M., Matsumoto, Y., and Hozumi, T. 1987. Human monoclonal antibodies to Pseudomonasaeruginosa produced by EBV-transfbrmed cells. Microbiol. Immunol. 31: 959-966. Terashima, M., Uezumi, I., Tomio, T., Irie, K., Okuda, T., Yokota, S., and Noguchi, H. 1991. A protective human monoclonol antibody directed to the outer core region of Pseudomonas aeruginosa lipopolysaccharide. Inject. Immun. 59: 1-6. Tsuchiya, S., Yokoyama, S., Yoshie, O., and Ono, Y. 1980. Production of diphtheria antitoxin antibody in Epstein-Barr virus induced lymphoblastoid cell lines. j. Immunol. 124: 1970-1976. Yabe, N., Matsuya, Y., Yamane, I., and Takada, M. 1986. Enhanced formation of mouse hybridomas without HAT treatment in a serum-free medium. In Vitro 22: 363-368, Young, L.S. 1972. Human immunity to Pseudomonasaeruginosa, II. Relationship between heatstable opsonins and type-specific lipopolysaccharides. J. Infect. Dis. 126: 277-287. Zweerink, H. J., Gammon, M.C., Hutchison, C.F., Jackson, J. J., Lombardo, D., Miner, K.M., Puckett, J.M., Sewell, T. J., and Sigal, N.H. 1988. Human monoclonal antibodies that protect mice against challenge with Pseudomonasaeruginosa. Infect. Immun. 56: 1873 -1879. (Received for publication, July 3, 1992; in revised form, September 28, 1992)