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

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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

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Establishment of stable cell lines producing anti-Pseudomonas aeruginosa monoclonal antibodies and their protective effects for the infection in mice.

Human-human hybridomas producing monoclonal antibodies (MoAbs) specific for five major serotypes of Pseudomonas aeruginosa were developed by fusing P...
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