Note: Descriptions are shown in the official language in which they were submitted.
oa~3s
HUl~AN MONOCLONAL ANTIBODY, AND ITS PRODUCTION AND USE
The present invention relates to a human mono-
clonal antibody to Pseudomonas aeruginosa (hereinafter
referred to as "P. aeruginosa"), and its production and use.
More particularly, it relates to a human monoclonal anti-
body, which has a specific binding property to flagella of
P. aeruginosa, and its production and use.
Noticeably, the human monoclonal antibody shows
its binding property not only to variou~ strains of
different serotypes based on O-an~igen but also to strains
belonging to the serotype having no O-antigen, i.e. Type M
according to the classification by Nippon Ryokunoh-kin
Kenkyukai Kesseigata-betsu Kento Iinkai (Commitee of Study
on Serotype Classification, Japanese Study Group on
Pseudomonas aeruginosa) (hereinafter referred to as
"Japanese Committee"~. Further, it exerts a remarkable
therapeutic effect on the experimental mouse infection
caused by P. aeruginosa at a dose of not less than 5 jug/kg
of body weisht. Thus, the human monoclonal antibody is
useful as a prophylactic or therapeutic agent on infectious
diseases caused by P. aeruginosa and also as a diagnostic
agent for such infectious diseases.
The kinds of bacteria causing infectious diseases,
i.e. infection-causing bacteria, have changed with develop-
ment of antibiotics as clinically used. As the result,
infections with bacteria originally having only low patho-
genicity or virulence have increased, and P. aeruginosa is
- 2 ~ 8 8 3~
currently one of the major pathogenic bacteria eausing
infectious diseases, of which serious symptoms often lead
patients to death, particularly when their immunologieal
activity is low due to continuous administration of immuno-
supressants or they are suffered from immunodeficieney or
immunodepression diseases eaused by caneer, burn or the
like.
~ nong various prophylactie or therapeutie methods
for baeterial infections, the most prevailing one is
chemotherapy by the use of antibiotics or antimicrobial
agents. In fact, there have been developed various anti-
biotics ineluding streptomycin, kanamycin, penieillin,
cephalosporin, ete., whieh are generally sensitive to
Gram-positive baeteria (e.g. Staphyloeoeei) and Gram-
negative bacteria (e.g. E. coli) and produee a prominent
elinieal effeet. ~owever, there are known only few medi-
einal products sensitive to P. aeruginosa, and even those
medieinal produets act on P. aeruyinosa only baeterio-
statically but not bacteriocidally. Thus, they can prevent
the growth of P. aeruginosa but do not exert any remarkable
therapeutic effeet in clinic.
, .~., ,y
Th~ other prophylaetie or therapeutic method is
antibody therapy by administration of immunoglobulin~ This
method is performed in place of or sometimes in assoeiation
with ehemotherapy. A serum of high antibody titer ean be
obtained by aetive immunization of animals sueh as horse or
rabbit, and antibody therapy ean be made through administra-
tion of queh serum. In fact, its remarkable therapeutie
2~(38~3~3
effect has been proven on experimental infections usiny
various animals. AS seen in the cases of diphtheria toxin
and viper toxin/ antibody therapy using sera originated from
animals is quite effective even for human beings. However,
introduction of a heterogenic protein obtained from animals
into a human body causes such a serious side-effect as
anaphylaxis or any other allergic reaction, so that the sera
originated from animals is not used therapeutically for
bacterial infections. It is thus highly desired to develop
human immunoglobulin having a high a~tibody titer against
bacteria and showing a prominent therapeutic effect on
bacterial infections.
Conventional human immunoglobulin preparations are
manufactured by collecting blood from healthy persons or
bacteria-infected patients, subjecting the blood to frac-
tionation to obtain an immunoglobulin fraction, purifying
the immunoglobulin fraction and eliminating therefrom
agglutinated materials through addition of ethylene glycol,
treatment with protease, sulfonization, DEAE column chroma-
tography, e~c., followed by formulation of the resulting
product into intramuscularly or intravenously injectionable
preparations. These prepara~ions are advantageous in not
causing anaphylaxis or any other side~effect as seen in ad-
ministration of immunoglobulin originated from animals but
yet have some drawbacks. One of such drawbacks is that
their antibody titer against bacteria is low, so that a
sufficient therapeutic effect can not necessarily be
produced. Another drawback is that their stable supply with
~g~
a, --
a high antibody titer in a large amount is dif~icult,
because they are prepared with the blood collected from
healthy persons or bacteria-infected patients. So, the
constant and continuous obtainmen~ of sera having a high
antibody titer is quite hard. A further drawback is that
they may be contaminated with hepatitis virus (e.g. HB
virus), Adult T cell leukaemia virus (ATLV, HTLV~, etc.,
since the blood as the starting material is obtained from a
number of persons. In order to avoid these drawbacks,
production of a human monoclonal antibody having a strong
prophylactic or therapeutic effect on bacterial infections
caused by P. aeruginosa is highly demanded.
On the surface layer of the bacterial body of P.
aeruginosa, there exist a large number of antigens, on which
many studies and researches have targetted so as to provide
a vaccine or antibody to be prophylactically or therapeu-
tically usable in treatment of infectious diseases caused by
P. aeruginosa. As the target antigen, there are known
O-antigens, outer membrane proteins, cilia (i.e. pili or
fimbriae3, flagellaj mucoids, etc.
The O-antigens are widely used for classification
of P. aeruginosa strains on their serotypes and correspond
mainly to differences in the construction (i.e. component,
sequence and configuration) of the repeating unit of the
O~polysaccharide portion in the lipopolysaccharide (LPS~
existing in the outer membrane. Typical examples of the
serotype classification are as follows: Types l to 17
according to the classification by Homma et al. (Japan. J.
38~3~
-- 5 ~
Exp. Med., 44, 1, (1974)); Types 1 to 7 according to the
classification by Fisher et al (J. Bacteriol., 98, 835
(1969)); Tvpes A to M according to the classification by
Japanese Committee (Japan. J. Exp. Med., 45, 329 (1976));
Types 1 ~o 17 according to the classification by Inter-
national Antigenic Typing System (IATS) lInt. J. Syst.
Bacteriol., 33, 256 (1983)), etc. These classifications and
their relationship are shown in Table 1 (Japan. J. Exp.
Med., 46, 329 (1976)~.
Table 1: Serotype classification of P. aeruginosa
, Japane~e ! Homma et alITATSFisher et al
i Commitee1974 1983 1969
', 1976 _ _
E2, 7, 13, 162, 5, 16 ¦ 3, 7
I 6 11 2
L15, 177, 12, 14, 17 ~ -
It is known that the antibody specific to a
certain O-antigen shows a skrong prophylactic or therapeutic
effect against P. aeru~inosa strains o~ the serotype to
which said O-antigen belongs but not against P. aeruginosa
strains of other serotypes. Further, even the antibody
effective to the O-antigen in any serotype is not effective
to Type M strains which do not possess any O-antigen. In
2~3~3~
-- 6
order for the antibody to be effective against P~ aeruginosa
strains of every serotype, it may he considered to use all
the monoclonal antibodies corresponding to those serotypes
in mixture. This technique is, however, eY.tremely trouble-
some and so far hardly applicable to the practical use.
Major outer membrane proteins are antigens common
to P. aeru~inosa strains. For instance, Hancock et al.
confirmed eight proteins (Dl, D2, E, F, G, Hl, H2 and I) as
the outer membrane protein and disclosed a part of their
characteristics (J. Bacteriology, 140, 802 (1979)~. Further
investigation using anti-serums against the outer membrane
protein revealed that the outer membrane proteins designated
as F, H2 and I could possibly be used as a vaccine (J.
Infec. Disease, 146, 770 (1982)). Based on these knowledge,
they succeeded in obtaining a mouse monoclonal antibody to
H2 and I proteins ~Infect. Immun., 37, 166 (1982)~ as well
as a mouse monoclonal antibody to F protein (Infec. I~nun.,
42, 1027 (1983)) and subsequently confirmed that the mouse
monoclonal antibody corresponding to F protein is effective
in treatment of the ln vivo experimental PO aeruginosa
infection iIl mice (Eur. J. Clin. Microbiol., 4, 224 (1985)).
Likewise, Sokol et al. reported tha~ ferripyochelin-binding
protein (FBP) is an antigen common to P. aeru~inosa strains
and a mouse monoclonal antibody to said FBP is effective in
treatment of the experimental P. aeru~inosa infection in
mice (Infect. Immun., 51, 896 (1986)). Nevertheless,
current studies and researches on the outer membrane
proteins have not sufficiently been developed, and un-
-- 7
clarification still remains on the characteristics, func-
tions, etc. of each outer membrane protein of P. aeruqinosa,
particularly on the relationship of such outer membrance
protein with the pathogenicity of P. aeru~inosa.
Cilia, flagella and mucoids existing on the
outside of the outer membrane are known to have a close
correlation with the pathogenicity of P. aeruginosa. Woods
et al., for instance, reported that cilia take part in the
adherence of P. aeruginosa onto the upper respiratory epi-
thelium, followed by colonization (Infect. Immun., 29, 1146
(1980)). Also, Ramphal et al. confirmed that said adherence
has a correlation to cilia and mucoids (Infect. Immun., 44,
38 (1984); ibid., 47, 1 (1985)). Further, McManus et al.
(Burns, 6, 135, 1980) and Carven et al. (Can. J. Microbiol.,
25, 458 (1981)) reported that a motility of P. aeruginosa
due to flagella is correlated to its pathogenicity. Further-
more, Montie et al. made comparison of toxicities in the
experimental infections of burned mice with P. aeru~inosa
strains having flagella and with their muta~ed strains
having no flagella and confirmed that fla~ella is the
important pathogenic factor (Infect. Immun., 38, 1296
(1982)). Moreover, Montie et al. reported that in the in
vitro experiment the anti-serum to flagella could suppress
the motility of P. aeru~inosa (Infect~ Immun., 35, 182
(198~)).
In addition, Holder et al. reported the effective-
ness of the prepaxation originated from flagella as a
vaccine in the experimental P. aeruginosa infections with
3 ~
burned mice (infect. Immun., 35, 220 (1932)). Drake e~ al.
showed the effectiveness of the anti-serum to flayella in
the experimental P. aeruyinosa infections with burned mice
(Can. J. Microbiol,, 33, 755-763 (1987)). Lostrom et al.
provided a hybridoma cell line capable of producing a mouse
monoclonal antibody to flagella of P. aeruginosa as well as
an EB virus transformed cell line capable of producing a
human monoclonal antibody to flagella of P. aeruginosa and
submitted an experimental report on the therapeutic effect
of those antibodies in the experimental P. aeruginosa
infections in mice (JP-A-63-102697). From their experi-
mental report, however, it is noted that the therapeutic
effect is obtainable only at a relatively high dose.
Namely, in case of the subcutaneous adminis~ration of the
human monoclonal antibody premixed with bacteria, the dose
of said antibody is to be 10 ~g per animal for exertion of
its therapeutic effect, and in case of the intravenous
administration of the human monoclonal antibody 2 hours
before the bacterial infection, the does is to be 40 ~g
~corresponding to 0.5 - 2 mg/kg) (cf. Examples 8, 10, 11 and
12 in JP-A-63-102697). Based on these experiments, the
possible effective dose for human beings would be about 1 to
200 mg/kg for the therapeutic purpose and about 0.1 to 25
mg/kg for the preventive purpose. Taking the currently
available technique for cultivation of the animal cells and
purification of the monoclonal antibody into consideration,
the mass production of the prophylactic or therapeutic agent
according to Lostrom et al. at a low cost will be difficult.
~g~3~
-- 10 ~
infectious diseases caused by P. _eru~inos~ at a rela~ively
low dose such as about 5 pg/kg per body weight or more.
Another object of this invention is to provide a human cell
line capable of continuously producing said human monoclonal
antibody and its descendant cell lines. A further object of
the invention is ~o provide a process for production of said
human monoclonal antibody by the ln vitro cultivation of
said human cell line. A still further object of the inven-
tion is to provide a human immunoglobulin preparation
comprising said human monoclonal antibody, which is usable
for prophylactic or therapeutic treatment of the infections
caused by P. aeruginosa.
A flagellum is a motile small organ and comprises
a basal body bound on th~ outer surface layer of the cell
body and a spiral fiber protruded therefrom with a hook
connecting them. In case of P~ aeruginosa, the spiral fiber
is made of a kind of flagellin, of which units are spirally
connected by hydrophobic bonding to form a cylindrïcal
structure. Flagellin is a granular protein, and that of P.
aeruginosa is readily obtainable by the Pitt et al. method
(J. Med. Microbiol., 14, 251 (1981)~ and can be purified
with ease according to the affinity column chromatography
using a human monoclonal antibody corresponding thereto.
As to the biochemical observation of flagella of
P. aeruginosa, Montie et al. reported that the apparent
molecular weight of flagellin constituting said flagella is
in a range of 53,000 to 45,000 when determined by sodium
dodecylsulfate-polyacrylamide gel electrophoresis (herein-
~g~,~8
-- 10 --
infectious diseases caused by P. aeruyinosa a~ a relativ~lylow dose such as about 5 /ug/kg per body weight or more.
Another object of this invention is to provide a human cell
line capable of contil~uously producing said human monoclonal
antibody and its descendant cell lines. A further object of
the invention is ~o provide a process ~or production of said
human monoclonal antibody by the ln vitro cultivation of
said human cell line. A s~ill further object of the inven-
tion is to provide a human immunoglobulin preparation
comprising said human monoclonal antibody, which is usable
for prophylactic or therapeutic treatm~n* of the infections
caused by P. aeru~inosa.
A flagellum is a motile small organ and comprises
a basal body bound on the outer surface layer of the cell
body and a spiral fiber protruded therefrom with a hook
connecting them. In case of P~ g~ , the spiral fiber
is made of a kind of flagellin, of which units are spirally
connected by hydrophobic bonding to form a cylindrical
structure. Flagellin is a granular protein, and that of P.
aeruginosa is readily obtainable by the Pitt et al. method
(J. Med. Microbiol., 14, 251 (1981)~ and can be purified
with ease according to the affinity column chromatography
using a human monoclonal antibody corresponding thereto.
As to the biochemical observation of flagella of
P. aeruginosa, Montie et al. reported that the apparent
molecular weight of flagellin constituting said flagella i5
in a range of 53,000 to 45,000 when determined by sodium
dodecylsulfate-polyacrylamide gel electrophoresis (herein-
3 ~
after referred ~o as "SDS-PAGE") ~Infec. Immun., 49, 770
(1985)). Further, Lanyi et al. reported that a thermolabile
antigen (i.e. H-antigen), which is often detected in ~he
agglutination method for classi~ication of P. aeruginosa, is
mainly correlated to flagella and established the classifi-
cation of flagella on the serotype (Acta Microbiol. Acad.
Sci. Hung., 17, 35 (1970)). Similar reports were also made
by Ansorg et al. (Zentralbl. Bakteriol. Parasitenkd.
Infektionskr. ~yg. Erste Abt. origO Reihe A Med. Mikrobiol.
Parasitol., ~42~ 228 (197833 and by Pitt et al. (J. Med.
Microbiol., 14, 251 (1981)). While the relationship of the
serotypes in these classifications are not clear, Pitt et
al. stated that Pitt H3 corresponds to Lanyi Hl and Ansorg
Hb and is a homogeneous serotype having no subgroup, and
Pitt Hl, ~2, H4, H5 and H6 correspond to Lanyi H2 and Ansorg
Ha and is a heterogeneous serotype having a subgroup.
The term "monoclonal antibody" herein used means
an antibody having a uniform molecular structure and being
produced by a single antibody-producing clone, which is
obtainable by cell fusion ~Nature, 256, 495 (1975)~, EB
(Esptein~Barr) virus transformation (Proc. Natl. Acad. Sci.
USA., 70 (1973)) or the like. The antibody is a glyco-
protein having a specific binding property to an antigen and
can be produced by B lymphocytes. It is classified into the
following groups. IgG, IgM, IgD, IgA and Ig~; for in-
stance, IgG has a molecular weight of about 150,000 and
comprises two heavy chains (H chains) having each a mole-
cular weight of about 50,000 and two light chains (L chains)
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- 12 ~
having each a molecular weiyht of about 25,000, each of said
H chains and L chains being covalently bound through a
disulfide residue and one molecule of IgG being bound
specifically to two molecules of antigen.
Bvaluation of the therapeutic effect of the human
monoclonal antibody in an experimental P. aeruginosa infec-
tious diseases in mice is made as follows: into groups of
mice received a designed amount of the human monoclonal
antibody ~i.e. medicaged group), various amounts of P.
aeru~inosa cells are inoculated, and the LD50 value ~the
amount of cells leading half the mice to death) and the
upper and lower limits of the LD50 value with 95 % confi-
dence limit are statistically determined; the same determi-
nation as above is also made on the non-medicated group or
the BSA (bovine sexum alubmin~ administration group for
comparison; when the LD50 value range of the medicated group
is not overlapped with that of the group for comparison, it
is taken as the presence of significant difference between
them, and the human monoclonal antibody is regarded to be
therapeutically effective.
The present invention will be further explained in
details below.
The human monoclonal antibody of the invention is
a homogeneous antibody of human type originated from a
single antibody-producing cell clone, which can recognize
specifically flagellin constituting the flagella of P.
aeruginosa as an antigenic determinant, has a specific
binding property to said flagella and produces a definite
- 13 ~
therapeutic effect in the experimental P. aeruyinosa infec-
tions with burned mice by intraveous administration at a
dose of 5 ~g/kg of body weight or more. This antibody is
quite characteristic in showing a binding property to a wide
variety of P. aeruginosa strains of different serotypes
based on O-antigen, particularly including Type M strains
having no O-antigen.
For instance, the antibody produced from Hybridoma
IM-2A8 shows a binding property to 50 to 65 ~ of the
clinically isolated strains of P. aeru~ belonging to
Type A (classified based on O-antigen), 50 to 60 % of those
in Type B, 40 to 50 % of those in Type H, about 30 ~ of
those in Type I and about 35 ~ of those in Type M without
any material binding property to those of Types E and G.
Thus, the antibody shows a binding property to 30 % or more
of all the clinically isolated s~rains including Types ~, B~
E, G, H, I and M. Due to such wide recognizability, the
antibody is usable for isolation and identification of P.
aeruginosa strains. In addition, the antibody specifically
recogni2es flagella or flagellin as its constitutent, which
correspond to Type Hb by the An~org et al. classification.
Accordingly, the human monoclonal antibody of the
invention may be used for prevention and treatment of a wide
variety of infectious diseases with P. aeru~inosa of differ-
ent serotypes based on O-antigen, particularly ircluding
Type M strains having no O-antigen by its administration at
a low concentration, which causes lysis of the bacterium
by a complement, accelera~ion of phagocytosis of a macro
3 g
phage and supression of the motility of bacterium, leading
to make P. aeruginosa less virulent.
The human monoclonal antibody o the invention
generally belongs to IgM but is not limited to said class.
For production of such antibody, there may be adopted a
method which comprises fundamentally the following stages:
(1) preparation of human B lymphocytes sensitized with an
antigen; (2) establishment of the cell line capable of
producing a specific monoclonal antibody by immortalization
of the B lymphocytes as prepared in (1); (3~ cultivation of
the cell line as established in (2); (4) purification of the
monoclonal antibody from the culture super~atant as obtained
in (3); and (5) production o~ an immunogloblin preparation
of high titer comprising the monoclonal an~ibody as purified
in (4). Each of these stages will be hereinafter
illustrated in details.
Stage (1):-
As the human B lymphocites, there may be usedhuman lymphocyte cells producing an antibody to ~lagella of
P. aeruginosa, which can be separated from a peripheral
blood by the centrifugation using a lymphocyte separation
liquid such as Lymphoprep~ or Mono-Poly Resolving Medium~
(Flow Lab.). There may be also used B lymphocytes origi-
nated from tissues or organs (e.g. lymph node, spleen)
extracted for the purpose of diagnosis or therapy of
diseases, umbilical cord blood or the like. It is desirable
to obtain the cells from persons once infected with P.
aeru~inosa and whose cells are sensitized by the infection.
~g8~
~ 15 -
Pertinent persons, from whom the cells may be obtained, can
be chosen by previous measurement of the antibody titer in
their sera to formalin fixed P. aeruginosa cells or flagella
of P. aeruginosa by the ELISA method or the radioimmunoassay
-
method. Alternatively, human B lymphocytes may be obtained
from any person irrespective of his or her past medical
history. Such lymphocytes are mixed with formalin-fixed P.
aeruginosa cells or preferably flagella of P. aeruginosa as
an antigen for ln vitro sensitization. Also, solutions
containing lymphokines such as B cell proliferation factors
and B cell differentiation factors (e.g. plant lectins such
as pokeweed mitogen (PWM), bacterial components such as
Cowan I, human mixed lymphocyte culture supernatant, spleen,
thymus or umbilical cord blood cell culture supernatant) may
be added to human B lymphocytes culture for sensitization in
vitro, followed by proliferation and differentiation to give
antibody-producing cells. These procedures such as pre-
selection and in vitro sensitization are effective to obtain
the spefic antibody-producing cell lines with a high effi-
ciency after cell fusion. The thus obtained human B lympho-
cytes characteristically have antibody molecules at the cell
surface and can release a certain amount of antibody for a
limited period of culture, but their proliferation over an
unlimited period of time is not possible.
Stage (2):-
For changing the above sensitized human B lympho-
cytes to continuously and perpetually proliferable cell
lines, the sensitized human lymphocytes are subjected to
~8536~
- 16 -
cell fusion with myeloma cells in the presence of poly-
ethylene glycol (PEG). The myeloma cells as used are
hypoxanthine-guanine-phosphoribosyl transferase (HGPRT~-
deficient mutants (e.g. P3X63-Ag 8(P3), P3X63-Ag 8.653)
originated from mouse myeloma cells, HGPRT deficient mutants
originated from mouse-human heteromyeloma cells obtained by
cell fusion between mouse myeloma cells and human myeloma
cells or between mouse myeloma cells and human lymphocyte B
cells, etc.
As the polyethylene glycol (PEG), there may be
used, for instance, PEG 1,000 to 6,000 in a concentration of
30 to 50 % (w/v). The fusion efficiency can be enhanced in
the presence of lectin, poly-L-lysine, dimethylsulfoxide
(DMSO), etc.
The fusion may be carried out, for instance, in
the same manner as described in the Kohler et al. article
(Nature, 256, 495 (1975)~ wherein mouse cells are fused each
other to obtain a hybridoma producing a mouse monoclonal
antibody. For instance, antigen-sensitizea human lympho-
cytes and HGPRT-deficient myeloma cells or human-mouse
heteromyeloma cells are mixed together in a proportion of 3
- 1 : 1, 45 % (w/v~ PEG 1500-6000 is added portionwise
thereto in a period of 0.5 to 1 minute, and the resultant
mixture is allowed to stand for 0.5 to 3 minutes. To the
resulting mixture, 10 to S0 ml of a culture medium contain-
ing no serum are added in 5 to 10 minutes, and subsequently
2 ml of FCS are added, followed by incubation at 37C for 10
to 60 minutes. After centrifugation, fresh culture medium
- 17 ~ 8~3~
is further added thereto to make a cell concentration o 105
to 106/ml. The ceil suspension thus obtained is seeded to a
96 well microplate at a rate of 2 x 104 to 2 ~ 105 cells per
well. On the next day, the half amount is replaced by a
hypoxanthine-aminopterin-thymidine~containing medium (HAT
medium) or a hypoxanthine-azaserine-containing medium (HAz
medium), and cultivation is done at 32 to 37C under 5 %
CO2. For about 10 to 20 days, the culture medium is re-
placed by fresh HAT medium or HAz medium, and subsequently
by hypoxanthine-thymidine-containing medium (HT medium~ or
hypoxanthine-containing medium (H medium~ for about 3 to 5
days, each replacement being made every 3 days for the half
amount over a period of about 2 to 3 weeks to obtain a
proliferative colony, i.e. hybridoma. It is also possible
to select a hybridoma by the combined use of metabolism
inhibitors without using a HGPRT-deficient mutant.
The antibody titer of the culture medium to
formaline-fixed P. aeruginosa cells or to flagella of P.
aeruginosa is determined by ELISA or radioimmunoassay (RIA),
if necessary, in combination with Western blotting to select
the desired cell capable of producing the specific antibody
to flagellin of P. aeruginosa. Cloning is repeated two or
three times by the limiting dilution method or the agarose
method to obtain a stable cell line having a high proli-
eration rate and a high specific antibody productivity.
The cell lines as established from sensitized
human B lymphocytes according to the cell fusion thybridoma)
method can be proliferated continuously, and in addition,
- la -
they can be cultivated in a medium containing no serum at a
large scale to give stably a large amount of the specific
antibody.
Stage (3) -
The thus established hybridomas (0.5 - 5 x 105
cells/ml) are cultured in a stationary or rota~ion manner in
a vessel (e.g. flask, plate) comprising a conventional cell
culture medium containing or not serum using a CO2 incubator
under 2 to 10 ~ CO2 at 32 to 37C. The conventional culture
medium may be, for instance, a medium (e.g. RPMI1640,
Eagle's MEM) containing 2 to 20 ~ ser~n of bovine fetus,
calf, cow, horse, human or the like, a serum-free medium
containing trace components required for the growth of cells
(e.g. insulin, transferrin, ethanolamine, selenite, bovine
albumin, lipid) or the likeD Particularly when culture is
made at a large scale, a jar fermenter, a hollow fiber
system or the like designed for animal cells may be used.
Stage (4):-
Purification of the antibody may be carried out byconventional biochemical procedures such as ammonium sulfate
precipitation, ethanol precipitation, PEG fractionation, ion
exchange chromatography, gel filtration, affinity chromato-
graphy, high performance liquid chromatography, electro-
phoresis, etc. In the purification process, care should be
taken for preventing the production of agglutination or the
depression of antibody activity. For this purpose, human
serum albumin (HSA) may be added in an amount of 0.05 to 2
~. Also, addition of amino acids such as glycine or alpha-
1 9
alanine, especially basic amino acids such as lysine,arginine or histidine, saccharides such as glucose or
mannitol, salts such as sodium chloride, etc. is occa-
sionally preferred. Agglutination tends to be produced,
particularly in the case of IgM antibody, and treatment with
beta-propionolactone, acetic anhydride or the like is
effective in prevention of such agglutination. In such
case, intravenous administration without any agglutination
will be made possible.
Stage (5):-
The purified monoclonal antibody may be formulatedinto a biological preparation by a ~ se conventional
procedure comprising, for instance, filtering through a
membrane filter for removal of bacteria and admitting into
sterilized vials with a stabilzer, followed by lyophily-
zation.
The human monoclonal antibody preparation thus
prepared may comprise only one kind of human monoclonal
antibody reactive to one antigenic determinant of flagellin
of P. aeruginosa for its use as a prophylactic or thera-
peutic agent for infections caused by P. aeruginosa.
Preferably, however, it comprises two or more kinds of human
monoclonal antibodies which can recognize different anti-
genic determinants of flagellin in P. ~ g~ . It may
also comprise additionally a human monoclonal antibody which
can recognize any of other surface layer antigens of P.
aeruginosa such as outer membrane proteins and O-antigens in
LPS, pathogenic factors of P. aeruginosa such as exotoxins,
c~ 3 g
- 20 -
exoenzymes such as elastase and protease, etc. or be admixed
with any conventional human immunoglobulin preparation for
them. It may be further admixed with any human monoclonal
antibody to bacteria other than PO aeruginosa, virus, fungi,
protozoa, tumor cells, etc. or any conv~ntional human
immunoglobulin preparation for them. Incorporation of the
human monoclonal antibody of the invention into a conven-
tional human immunoglobulin preparation makes a high titer
immunoglobulin preparation to P. aeruginosa. For mixed
bacterial infections, the human monoclonal antibody of the
invention may be applied in combination with any conven-
tional antibiotic or synthetic antimicribial agentO
The human monoclonal antibody of the invention
recognizes specifically flagellin and binds to flagella of
P. aeruginosa, whereby the bacterial body is opsonized and
the phagocytosis and bacteriolysis actions of phagocytes
thereon are enhanced. Also, complements are activated to
accelerate the lysis of the bacterial body~ Further, the
motility of P. aeruginosa is suppressed to lower the patho-
genicity of the bacterium. Due to these actions, the human
monoclonal antibody of the invention produces a significant
therapeutic effect in the experimental mouse infections with
P. aeru~inosa even at such a small dose as 5 ~g/kg. In view
-
of this experimental result, the human monoclonal antibody
of-the invention may be administered to an adult human
patient in an amount of about 0.05 to 1 mg per kg of body
weight for the therapeutic purpose of infectious diseases
with P. aeruginosa alone or mixed bacterial infection
3 ~
- 21 -
20containing P. aeru~lnosa or in an amount of about 0.005 to
-
0.1 mg per kg of body weight for the prophylac~ic purpose.
As stated above, the human monoclonal antibody has
a specific binding property to flagella of P. aeruginosa and
exerts its therapeutic effect on the experimental mouse
infections caused not only by various strains having
O-antigen but also by strains having no O-antigen (Type M1.
This is quite noticeable~ because any monoclonal antibody to
flagella having material therapeutic effect on infections
caused by Type M strains is never reported and of course
monoclonal antibody to O-antigen has no effect. This is
also noticeable, because Type M strains are isolated as the
infection-causing organism no~ only from ordinary infectious
diseases caused by P. ~ with a frequency of several
percent but also from cystic fibrosis and DPB known as quite
serious diseases with a significantly high frequency.
In addition, it may be noted that the therapeutic
effect of the human monoclonal antibody of the invention can
be enhanced prominently when this is used in combination
with conventional antibiotics or synthetic antimicrobial
agents sensitive to P. aeruginosa, and this enhancement is
significantly synergistic. This prominent synergistic
effect is presumed to arise from the mechanism that the
human monoclonal antibody not only opsonizes the bacterium
to induce phagocytosis but also suppresses the motility of
bacterium to ma~e P. aeruginosa less virulent. This may be
considered to be a characteristic feature inherent to the
human monoclonal antibody according to the in~ention which
7J~(~8~3~
~ 22 -
specifically binds to flagella of P. aeruginosa.
Currently, most of serious P. aeruginosa infec-
tious diseases are provoked by mixed bacterial infections
containing P. aeruginosa. Therefore, theraphy of those
diseases includes the application of conventional chemo-
therapeutic agents such as antibiotics and synthetic anti-
microbial agents in combination with human immunoglobulin
preparations comprising the human monoclonal antibody.
Since enhancement of the synergistic effect in the combined
use of the human monoclonal an~ibody with the conventional
chemoterapeutic agents i5 prominent as noted above, it is
quite advantageous for prevention and treatment of serious
P. aeruginosa infectious diseases caused by mixed bacteria
including P. aeruginosa.
-
As understood from the above, the human monoclonalantibody of the inven~ion has various meri~orious advan-
tages. First, it binds specifically to flagella of PL
aeruginosa and shows an excellent therapeutic effect in the
experimental mouse infections at such a small dose of not
less than 5 ~g/kg o~ body weight. Since the effective dose
for prevention or treatment of the infectious diseases is
small, the immunoglobulin prepration comprising the human
monoclonal antibody can be supplied with a low cost.
Second, the human monoclonal antiboby shows its binding
property to a wide variety of PO aeruginosa strains includ-
ing Type M strains, and therefore its therapeutic effect is
exerted on khe infectious diseases caused by P. ~
strains of different serotypes. Thus, it is possible to
g
- 23 -
avoid any inconvenient operation for mixing a plurality of
monoclonal antihodies, yet achieving a therapeutic effect on
the infectious diseases caused by different serotypes of
strains. Third, the human monoclonal antibody of the
invention is a human origin protein so that any side effect
(e.g. anaphylaxis) as seen in the administration o~ a
heterogenic protein is hardly produced. Since it is origi-
nated from a certain specific cell line, a possibility of
contamination with unknown biohazardous materials is much
less in comparison with conventional immunoglobulins
prepared from a human blood originated from a number of
persons.
The antibody-producing cell line of the invention
is characteristic in showing a high proliferation and a
significant antibody productivity continuously at a large
scale cultivation. The large scale cultivation is possible
even in a serum-free medium and stably affords the antibody
in a large amount. For instance, the large scale cultiva-
tion with the human-mouse heterohybridoma cell line IN-2A8
in a 6 liter-volume jar ermenter shows yood proli~eration
with high productivity of the antibody.
These good proliferation and high productivity of
IN-2A8 are very important for the industrial large scale
culture, so that clone IN-2A8 is a very useful cell line.
Characteristically, the binding property of the IgM antibody
produced from said cell line IN-2A8 to flagella of P.
aeruginosa is varied with the Ca ion concentration, the
maximum binding property being in a concentration of 100
~ 24 -
and the minimum (i.e. almost no) binding property being in a
concentration of 1 ~M or less.
According to the present invention, the human
monoclonal antibody is thus artificially and stahly produced
with a high antibody tit~r in a large amount. The produc-
tion process is more advantageous than conventional produc-
tion processes using human blood with respect to quality
control.
The present invention will be hereinafter ex-
plained in details by way of examples, but it should be
understood that this invention is not limi~e~ ~o t-hose
examples.
Example 1
~ stablishment of human an~i-flagellin monoclonal
antibody-producing cell lines by human~mouse cell fusion:-
1) Preparation, cultivation and activation ofhuman B lymphocytes
Peripheral blood (100 ml) having a high antibody
titer to _. aeruginosa (formalin-fixed cells) in serum was
taken from a healthy volunteer Idonor, IN). To a centrifuge
tube (50 ml-volume, Sumitomo Bakelite), there was admitted
Mono-Poly Resolving Medium~ (15 ml, Flow Lab.), and peri-
pheral blood (20 ml) was slowly overlaid thereon, followed
by centrifugation with a lo~ speed centrifuge (BS-20BH,
Tommy Precision Ind.) at Z,500 rpm (Roter-qlS-7) and at room
temperature for 15 minutes, whereby erythrocytes and lympho-
cytes were separated.
The portion containing lymphocytes was collected
- 25 ~ 8 ~ 3g
and washed three times with a Dulbecco's modified Eagle's
minimum essential medium (Nisshi Pharmaceu~ical, hersinafter
referred to as "D-MEM" ), followed by calcula~ion of the cell
numbers to obtain lymphocyte cells of 1.2 x 103.
The lymphocyte cells (1.2 x 108~ were su~pended in
a lymphocyte-culturing medium (60 ml) containing formalin-
fixed cells of P. aeruginosa, and the suspension was
dispensed in 24 well microplates (Costar, X3424) at a rate
of 2 x 106 lymphocy~e cells/well and cultured at 37C under
5 % C2 for 6 days. As the lymphocyte-culturing medium,
there was used an RPMI-1640 medium (Nissui Pharmaceutical)
containing inactivated fetal calf serum IFCS) (20 ~ (v/v)),
sodium pyruvate (0.05 mg/ml), 2-mercaptoethanol (5 x 10 5
M), N-(2-hydroxyethyl)piperazin-N'-2-ethanesulfonic acid
(hereinafter referred to as "HEPES") (20 mM) and pl~nt
lectin derived from pokeweed (PWM, Gibco Lab.) (0.01 ~
(v/v) ) .
(2) Cell fusion
Mouse myeloma cells P3x63-Ag8.653 (~TCC No.
CRL1580; J. Immunol., 123, 1548 (1979)) were subcultured in
D-MEM containing 10 % FCS, and 4 x 107 cells were washed
twice with D-MEM.
Separately, peripheral blood lymphocytes were
cultured for 6 days in 24 well microplates according to
Example l~tl) and recovered to give 8 x 107 lymphocyte
cells. The cells were washed with D-MEM three times and
mixed with the above mouse myeloma cells in a centrifuge
tube, followed by centrifugation. To the precipitate in the
~8~8
- 26 -
centrifuge tube while ro~ation, thexe was added a poly-
ethyleneglycol (PEG) solution tl ml) containing PEG4000
(Merck) (0.45 g~, PBS(~) (0.45 ml) and dimethylsulfoxide
(0.1 ml) in about one minute, and the mixture was allowed to
stand at room temperature for one minute. D-MEM was then
added to the tube at a rate of 2 ml/minute while rotation,
which was repeated seven times. Finally, 2 ml of FCS was
added to the tube, and the mixture was allowed to stand for
20 minutes at 37C. The cells were collected by centrifu-
gation and suspended in 50 ml of a D-MEM medium containing
FCS (15 %), sodium pyruvate (0.05 mg/ml~, insulin t0.2
U/ml), oxaloacetic acid (0.15 mg/ml), azaserine (100 ~M) and
hypoxanthine (100 /uM) (hereinafter referred to as "HAz
selection medium"). The suspension was dispensed in 96 well
microplates (#25860 MP, Corning Glass Works) at a rate of 8
x 104 myeloma cells/well. Simultaneously, each of the
microplates was charged with mouse BALB/c spleen cells (1 x
105) and mouse BALB/c peritoneal exudate cells i6.5 x 104)
as the feeder layer, and incubation was made at 37C under 5
% CO2. The half of the culture medium was replaced by the
HAz selection medium every 2 or 3 days. After one week, the
half of the culture medium was replaced by an H-medium which
corresponds to the HAz selective medium but excluding
azaserine therefrom. Thereafter, the half of the culture
medium was replaced by an azaserine and hypoxanthine-free
hybridoma-culturing D-MEM medium containing FCS ~15 ~,
sodium pyruvate (0.05 mg/ml), insulin (0.2 U/ml) and
oxaloacetic acid (0.15 mg/ml) every 2 or 3 days.
~ 27 ~ 8~3~
About 3 weeks after the cell fusion, production of
the antibody to the antigen at the surface layer of P.
aeruginosa surface was de~ermined on the supernatan~s of the
wells where proliferation was observed, by enzyme linked
immuno sorbent assay (ELIS~) using 96 well microplates
(#3912F, Bector Dic~inson Lab.) on which P. aeruginosa was
fixed by glutaraldehyde. In two wells, production of the
IgM antibody showing a strong binding property to P.
aeruginosa IID1002 (the standard s~rain of Type 2 according
to Homma et al. classification, obtainable from ATCC or from
the Institute of Medical Science, Tokyo University, Japan)
was confirmed. The heterohybridoma in these wells was
further cultivated in 96 well microplates and cloned by the
limiting dilution method under the condition giving a
uniform cell in each well to obtain heterohybridoma cell
lines IN-2A8 and IN-5D6 respectively having stable produc-
tivity of human monoclonal antibodies IN-2A8 and IN-5D6.
Hybridoma IN-2A8 was deposited under an accession No. FERM
P-9960 on March 24, 1988 at Fermentation Research Institute,
Agency of Industrial Science and Technology, located at
Tsukuba~ Ibaraki~ken, Japan, and this deposition was
converted into an internatinal deposition under Budapest
Treaty on January 22, 1990 under an accession No. BP-2741.
Example 2
Establishment of human anti-fla~ellin monoclonal
antibody-producing cell lines by human-mouse cell fusion:
Peripheral blood (100 ml) having a high antibody
titer to P. aeru~inosa (formalin-fixed cells) in serum was
- 28 - ~a~8~3~
taken from a healthy volunteer (donor, ZI), and preparation,
cultivation and activation of human B lymphocytes as well as
cell fusion were carried out in the same manner as in
Example 1 ~o give a human-mouse he~erohybridoma cell line
ZI-3A8 capable of producing the IgM antibody showiny a
strong binding property to P. aeruginosa IID1002, i.e. a
human monoclonal antibody designated as "ZI-3AB".
Example 3
Binding property of hwman IgM monoclonal anti-
bodies IN-2A8, IN-5D6 and ZI-3A8 to standard strains of P.
aeruglnosa:-
The binding properties of the antibodies IN-2A8,
IN-5D6 and ZI-3A8 to the standard strains of P. aeruginosa
according to the ~omma et al classification (obtained from
the Institute of Medical Science, Tokyo University, Japan)
were examined by ELISA, and the results are shown in Table
2, from which it is uders~ood that the antibodies IN-2A8,
IN-5D6 and ZI 3A8 show a strong binding property to IID1002
tType 2), IID1009 (Type 9), IID5141 (Type 14), IID5004 (Type
16) and IID1015 (Type 171, and their binding spectra are
quite similar to each other.
~8~3~
- 29 -
Table 2: Binding property of antibodies IN-2A8,
IN-SD6 and ZI~3A8 to standard strains
of P. aeruginosa
Strains Homma et al Japanese ELISA value (O~405)
Classifica- Commitee
tion IN-2A8 IN-5D6 ZI-3A8
. ___ _
IID1001 1 A 0.19 0.03 0.04
IID1002 2 B 0.97 1.99 1.60
IID1021 3 C 0.20 0.04 0.01
IID1004 4 D 0.16 0.00 0.00
IID1130 5 E 0.18 0.00 0.02
IID1006 6 F 0.17 0.00 0.00
IID1007 7 B 0.20 0. 06 0.01
IID10 20 8 G 0.1 6 0.00 0.00
IID1009 9 H 2. 07 2.23 2. 29
IID1010 10 I 0.04 0.00 0.00
IID1011 11 J O.OS 0.00 0.03
IID1012 12 K 0.04 0.00 0.00
IID1013 13 B 0.20 0.02 0.01
IID5141 14 L 0.53 0.45 0.52
IID5018 15 M 0.05 0.00 0.00
IID5004 16 B 0.76 1.75 1.54
IID1015 17 M 0.57 0.80 0~73
(-) ~ 1 0 04 ~ 05
Binding property of antibidies IN-2A8, IN-5D6 and
ZI-3A8 to clinical isolates of P. aeruginosa:-
The binding properties of the antibodies IN-2A8,
IN-5D6 and ZI-3A8 to P. aeruginosa clinical isolates Types
A, B, E, G, H, I and M (according to Japanese Commitee
classification) were examined by ELISA, and the results are
shown in Table 3-, from which it is understood that the
antibodies IN-2A8, IN-5D6 and ZI-3A8 show binding properties
to 58 ~ of Type A, 55 ~ of Type B, 30 % of Type I, 46 % of
Type H~and 35 % of Type M and no binding property to Types E
and G, and their binding spectra are quite similar to each
other. Thus, these antibodies may be considered to re~
~8~3~
- 30 -
cognize iden~icial antigens, which have certain correlations
to the serotype classification on O-antigens but are not
exactly identical therewith.
Table 3: Binding property of a~tibodies IN-2A8,
IN-5D6 and ZI-3A8 to P. aeru~inosa
clinical isolates
Strains Serotype ELISA value (OD405)
IN-2A8 IN-5D6 ZI-3A8
SP 6745 A 0.08 0.05 0.05
SP 6746 A 1.70 1.33 1.77
SP 6783 ~ Q.05 0.05 0.07
SP 6818 A 1.93 1.56 1.94
SP 6830 A 1.12 0.82 1.29
SP 6840 A 0.12 0.06 0.05
SP 9708a A 1.54 1.22 1.62
SP 9710 A 1.98 1~72 1.95
SP 9711 A 1.47 1.31 1.80
SP 9731 A 0.07 0.04 0.05
SP 9762 A 0.07 0.03 0.10
SP 9763 A 0.06 0.05 0.05
SP 9768 A 1.59 1.25 1.94
SP 9780 ~ 1.83 1.46 2.90
SP 10029 A 1.50 1.09 1.72
SP 10040 A 0.41 0.36 0.51
SP 10060 A 0.71 0.54 0.96
SP 10648 A 0.03 0.02 0.23
SP 10676 _ _ 0.05 0.05 0.04
SP 9703 B 0.07 0.04 0.04
SP 972? B 1.73 2.21 0.04
SP 9737a B 1.69 1.87 2.55
SP 9756 B 0.02 0.07 0.05
SP 9770 B 0.03 0.03 0.05
SP 9791 B 0.05 0.05 0.08
SP 10036 B 1.27 1.48 2~27
SP 10052 B 1.80 1.83 2.58
SP 10065 B 0.76 0.71 1.01
SP 10069 B 1.02 0.90 1.38
SP 10650 B 0.03 0.03 0.02
SP 10663 B 0.03 0.03 0.03
SP 10658 B 0.06 0.04 0.02
SP 10680 ~ 0.08 0.05 0.07
SP 6897 B 1.76 1.80 2.39
PAO 1 B 2.03 1.98 2.31
M 2 B 2.03 1.73 2.52
TL 2423 ~ 0.02 0.03 0.07
TL 2460 B 0.17 0.14 0.16
SP 6747 B 1.60 1.56 2.36
?~87~
- 31 ~-
(Continued)
SP 9702 0.06 0.06 0.00
SP 9715 E 0.10 0.10 0.00
SP 9720 E 0.07 0.07 0.00
SP 9723 E 0.10 0.10 0.08
SP 9726 E 0.06 0.06 0.00
SP 9733 E 0.05 0.05 0.01
SP 9740 E 0.05 0.05 0.00
SP 9754 E 0.07 0.07 0.01
SP 9759 E 0.07 0.05 0.03
SP 9771 E 0.05 0.05 0.00
SP 9783 E 0.06 0.05 0.00
SP 9787 E 0.05 0.05 0.00
SP 9790 E 0.05 0.05 O O OO
SP 10043 E 0.05 0.05 0.00
SP 10059 E O . OS 0.04 0.00
PA 103 E 0.05 0.06 0.00
PA 103-29 E 0.07 0.05 0.01
NC 5 E 0.06 0~04 0.00
TL 2092 E 0.08 0.06 0.02
TL 2158 E 0.06 0.06 0.02
SP 9701 0.00 0.00 0.00
SP 9709 G 0.00 0.00 0.00
SP 9712 G 0.00 0.00 0.00
SP 9714 G 0.00 0.00 0.30
SP 9717 G 0.00 0.00 0.00
SP 9718 ~ 0.00 0.00 O O OO
SP 9738 G 0.00 0.00 0.00
SP 9785 G 0.00 0.00 0.00
SP 9743 G 0.00 0.00 0.00
SP 9792 G 0.00 0.00 0.00
SP 9755 G 0.00 0.00 0.00
SP 9761 G 0.00 0.00 0.00
SP 9767 G 0.00 0.00 0.00
SP 9772 G 0.00 0.00 0.00
CTN11187 G 0.00 0.00 0.00
TL 2378 G 0.00 0.00 0.00
TL 2424 G 0.00 0.00 0.00
SP 6788 G 0.00 0.00 0.00
SP 9728a G 0.00 0.00 0.00
SP 6896 H 0.61 1.29 1.30
SP 6931 H 0.08 0.00 0.00
SP 7503 H 0.08 0.00 0.00
SP 7507 H 0.08 0.00 0 ~ 02
SP 7514 H 0.08 0.00 0 ~ 01
. SP 7520 H 0.08 0.02 0.05
SP 7522 H 1.08 1.97 1.94
SP 753~ ~ 1.18 2.03 2.16
SP 7555 H 0.08 0.02 0.01
SP 10054 H 0 O 08 0.00 0.00
SP 10068 H 0.93 1.92 1.97
SP 10678 H 1.16 2.14 2.15
SP 10681 . 0.45 0.98 1.20
- 32 - ~8~3~
(Continued)
_ .
SP 6761 I O O OO 0.00 0.04
SP 6722 I 0.00 0.00 0.04
SP 6805 I 0.00 0.00 0.05
SP 6811 I 0.39 0.36 0.32
SP 6829 I 0.00 0.00 0 ~ 03
SP 6839 I 0.27 0.28 0.31
SP 6853 I 0.35 0.27 0.30
SP 6880 I 0.00 0.00 0.03
SP 9704 I 0.00 0.00 0.04
SP 9705 I 0.00 0.00 0.03
SP 9706 I 0.00 0.00 0.06
SP 9707 I 0.00 0.00 0 O 07
SP 9735 I 0.14 0.15 0.22
SP 9757 I 0.00 0.00 0.03
SP 9776 I 0.00 0.00 0.03
SP 9779 I 0.00 0.00 0.04
SP 9799 I 0.00 0.00 0.06
SP 10041 I 0.00 0.00 0.05
SP 10046 I 0.71 0.68 0 ~ 67
SP 10570 I 0.27 0.26 0.29
_ . _ . . _ .
SP 9716 M 0.06 0.03 0.08
SP 9730 M 0.73 0.57 0.81
SP 9744 M 0.05 0.04 0.04
SP 9748 M 1.16 1.00 1.33
SP 9749 M 0.01 0.02 0.01
SP 9752 M 0.11 0.09 0.10
SP 9775 M 0 ~ 01 0.01 0.01
SP 10067 M 0.01 0.01 0.01
SP 10675 M 0.02 0.02 0.02
SP 6763 M 1.66 1.36 1.61
SP 6764 M 1.82 1.68 1.90
SP 6765 M 1.64 1.46 1.74
SP 6782 M 0.03 0.01 0.01
SP 6794 M 0.01 0.01 0.01
SP 6833 M 0.04 0.03 0.02
SP 6852 M 1.51 1.55 1.57
SP 6890 M 0.04 0.03 0 ~ 02
SP 68g2 M 0.82 0.72 0.94
SP 6895 M 0.01 0.01 0.01
SP 6908 M 0.01 0 01 0.01
_ _ _ I
(~) 0.01 1 0.04 0.02
- 33 ~ 8~38
Exam~le S
Antigen to be recognized by antibody IN-2A8:-
(1) Preparation of crude flagella product from P.aeruginosa
The crude preparation of flagella of P. aeru~inosa
was prepared by the method as described by Pitt et al (J.
Med. Microbiol., 14, 251 (1981)). P. aeruginosa strain M2
(Serotype B according to the Japanese Commitee classifica-
tion~, obtained from Shrinens Burns Institute, Cincinnati,
Ohio, U.S.A. (J. Infect. Dis., 131, 688 (1975)) and clinical
isolate strain SP 6818 (Serotype A), which shows a binding
property to the IgM antibody produced by Hybridoma IN-2A8,
IN-5D6 or ZI-3A8 in ELISA, were used.
Each of the above strains was cultured on a heart-
infusion agar medium (Nissui Seiyaka) for 18 hours. The
bacterial cells were scraped from the agar surface and
suspended in potassium and magnesium-free, phosphate-
buffered saline (hereinafter referred to as "PB5(-)") (10
ml), followed by centrifugation at 5000xg at 4C for 15
minutes. The precipited bacterial cells were again
suspended in PBS(-) (5 ml) and mixed by the aid of a Vortex
mixer for 3 minutes to release flagella from the bacterial
cells. Centrifugation was continued at 16000xg at 4C for
15 minutes, and a~ter remoYal of the bacterial cells, the
supernatant was further centrifuged at 40000xg at 4C for 3
hours to precipitate flagella. The precipitants were
suspended in PBS(-) (400 ~l~ to obtain a crude flagella
preparation.
~8~3~
- 34
(2) Electrophoretic analysis of the crude
flagella preparation on SDS-polyacrylamide gel
The crude flagella preparation ob~ained in (1) and
a molecular weight marker were mixed with a sample buffer
(50 pl) comprising tris buffer (6.25 mM; pH 6.8), SDS (2 %),
2-mercaptoethanol (10 ~), glycerol (10 ~) and BPB (0.001 ~),
and the mixture was heated at 95~C for 5 minutes and
subjected to electrophoresis on 10 ~ polyacrylamide gel
containing 0O2 ~ SDS and staining with Coomassie Brilliant
Blue ~ (Bio-Lad.). For the crude flagella preparation
originated from strain M2 or strain SP 6818, a major band
was recognized at the position of about 52,000 in molecular
weight, with a slight amount of impurities at the position
of about 60,000 in molecular weight. The above molecular
weight of about 52,000 for the crude flagella preparation is
almost identical to the molecular weight of 53,000 reported
for strain M2-originated flagelin by Montie et al (Infec.
Immun., 35, 281 (1982))~
(3) Binding property of the IgM antibody produced
by Hybridoma IN-2A8 as examined by western blotting method
Western blotting IProc. Natl. Acad. Sci., U.S.A.,
76, 3116 (1979)) was conducted to examine the binding
property of the IgM antibody to the crude flagella prepara-
tion. Namely, the crude flagella preparation as obtained in
(1) was subjected to electrophoresis in the same manner as
in (2) and then electrically transferred onto a poly-
vinylidenedifluoride membrane (Durapore~, Japan Millipore).
On the membrane, it was recognized by the enzyme immuno-
2 ~
- 35 -
staining method that ~he human monoclonal antibody IgM
produced by hybridoma IN-2A8 was bound to the major band
corresponding to flagellin having ~ molecular weight of
52,000, as shown in (2), in the crude flagella preparation
originated from strain M2 or SP 6818.
Still, the crude flagella preparation was treated
with a protease (Proteinase K~ at 37C for 2 hours and then
subjected to western blotting. As the result, it was
confirmed that the crude flagella preparation lost the reac-
tivity with the IgM antibody produced by Hybridoma IN-2A8.
From the above results, it is understood that the
IgM antibody produced by hybridoma IN-~A8 specifically
recognizes a granular protein constituting flagella of P.
aeruginosa, i.e~ flagellin, and is bound to the flagella.
Likewise, the IgM antibodies produced from ~Iybridomas IN-5D6
and ZI-3A8 can be bound to flagellin.
In summary, the IgM antibody produced by Hybridoma
IN-2A8, IN-5D6 or ZI-3A8 was confirmed by the western
blotting method to show a binding property to the strain
M2-originated flayellin. It was also confirmed by the ELISA
method that said antibody shows a binding property to the
strains M2 and PAO-1 (ATCC2524~ of P. aeru~inosa. Since
both of these strains are classified into Serotype Hb
according to the classification of Ansorg et al. on flagella
(Allison, Infec. Immun., 49, 770 (1985)), the IgM antibodies
produced from Hybridomas IN-2A8, IN-5D6 and ZI-3A8 may be
considered to have a specific binding property to Serotype b
flagella according to the classification of Ansorg et al.
- 36 -
and its constituent flagellin.
Example 6
Therapeutic effect of Antibody IN-2A8 on the
experimental P. aeruginosa infection in mice (1):-
ICR strain mice (4 week old; male; 10 animals pergroup; purchased from E~perimental Animal Farm Cooperation,
5hizuoka-ken, Japan) were shaved on the back, anesthetized
and forced to have a burnt on the back with pressing a burnt
ethanol-impregnated glass fiber thereto for 10 seconds. In
order to relieve the burnt shock, physiological saline (0.3
ml~ was intraperitoneally administered to each mouse, and
then a physiological saline suspension of P. aeruginosa M2
strain (O-antigen serotype B) or clinical isolate strain
SP 10052 (O-antigen serotype B) having a designed bacterial
concentration (0.2 ml) was subcutaneously administered at
the burn site for infection. After 1 hour, the human
monoclonal antibody IgM produced from Hybridoma IN-2A8,
being diluted with PBS(-) to ma~e a designed concentration,
was intraveneously injected (0.2 ml) to each mouse. After 7
days, the survival rate was observed to determine the
therapeutic effect. The results are shown in Table 4, from
which it is understood that the human monoclonal antibody
IgM originated from Hybridoma IN-2A8 clearly shows an
increase of the survival rate either at a dose of 0.1
~g/head or 1 ~g/head in comparisown with the non-medicated
group.
The LD50 value as well as its upper and lower
limits with a 95 ~ confidence limit for each dose were
~8~8
- 37 -
statistically calculated from the survival rate as shown in
Table 4, and the results are shown in Table 5, from which it
is understood that Antibody IN-2A8 shows a high LD50 value
either at a dose of 0.1 ~g/head or 1 ~g/head in comparison
wit the non-medicated group or the BSA-treated group. It is
also understood that the range between the upper and lower
limits of the LD50 value with the 95 % confidence limit for
the medicated group was not overlapped with that for the
non-medicated group or that for the BSA-treated group, and
the difference is thus significant. In other words, Anti-
body IN-2A8 may be understood to exert a definite thera-
peutic effect either at a dose of 0.1 ~g/head or 1 jug/head.
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Table 5~1: LD5 value in experimental infection
caused by M2 strain of P. _erug_nosa
in mice
.. ~ .
Antibody DoseLD50 value (95 ~ confidence limit)
(~g/head)
_ . 5 5 ~ 5)
IN-2A8 0.14.3 x 10 (1.3 x 10 - 14 x 10
BSA 0.15-7 x 103 (1 2 x 103 ~ 29 x lO
Non- _ < 1.3 x 103
medicated
. _ . _
Table 5-2: LD value in experimental infection
ca5~ed by clinical isolate (SP 10052)
of _. aeruginosa in mice
Dose of IN-2A8 LD50 value (95 ~ confidence limit)
(~g/head~ 3 2 4
0.01 1.2 x 10 (1.3 x 10 - 1.1 x 10
0.1 1.0 x 105 (2.2 x 104 - 4.5 x 1051
1 2.8 x 106 (1.2 x 106 - 6.5 x 106)
Non- 2.6 x 102 (4.9 - 1.5 x 104)
medicated
_ _ _
Example 7
Therapeutic effect of Antibody IN-2A8 on
experimental P. aeruginosa infection in mice (2):-
In the same manner as in Example 6 but usingdifferent serotype strains of P. aeruginosa, the therapeutic
effect of Antibody IN-2A8 was examined through the survival
rate and the LD50 value at a dose of 1 ~ug/head as shown in
Table 6.
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3 8
- 47 -
As understood from the above results, Antibody
IN-2A8 shows a therapeutic effect on the infection caused by
various O-antigen serotype strains of P aeru~inosa.
Further, the range between the upper and lower limits of the
LD50 value with the 95 ~ confidence limit is not overlapped
with that of the non-medicated group, and the difference is
thus significant. It follows therefrom that the treatment
with An~ibody IN-2A8 at a dose of l ~g/head has a signifi-
cant therapeutic effect.
Example 8
Large scale cultivation of Hybridoma IN-2A8 with
jar fermenter:-
~ ybridoma IN-2A8 was cultured in a jar fermenter
(6 liter-volume, Model MCT-3S, Marubishi Bioengineering)
using a commercially available serum-free culture m~dium
(Cellgrowther ~ Meguro Lab.) 14.4 liters) containing
glucose (final concentration, 4 mg/ml3 and FCS (2 %).
Hybridoma IN-2A8 was inoculated therein at a concentration
of l x 105 cells/ml and cultivated for 3 days to make a
concentration of 4.l x lO cells/ml. The concentration of
the human monoclonal antibody in the supernatant reached to
5.3 jug/ml. Every 2 to 3 days, stirring was intermittently
stopped to precipitate the calls, followed by replacement of
the upper supernatant containing no cell with a fresh
medium. With continuous cultivation in this way, a high
concentration of more than l x 106 cells/ml could be
maintained, and the concentration of the human monoclonal
antibody in the supernatant reached to lO ~y/ml at that
~8~g
- 4B -
stage.
Example 9
Purification of Antibody IN-2A8:-
In the same manner as in Examle 8, large scalecultivation of ~ybridoma IN-2A8 was performed in a jar
fermenter (6 liter-volume) using the Cellgrowther ~ medium
containing FCS (3 %). The supernatant (5.5 1) was
ultra-condensed to 100 ml (protein content, 2.6 g) and
charged on an Q-sepharose FF (Pharmacia, 2.6 cm (diameter) x
30 cm (length), 150 ml volume) equilibrated with PBS(-),
followed by washing with five times volume of 0.15 M
phosphate-buffer (pH, 6.8) and eluting with 0.3 M phosphate-
buffer to obtain IgM fractions. The fractions were
subjected to ultracondensation and charged on sephacryl
S300HR~ (Pharmacia, 2.6 cm (diameter) x 90 cm (length), 450
ml volume), followed by elution with PBS(-). The anti-
flagella antibody titer of each fraction was determined on
ELISA, and the active fractions were recovered. The
recovery rate of the human monoclonal IgM antibody as
protein was 1.2 %, and its specific activity raised to about
60 times.
Example 10
Therapeutic effect of Antibody IN-2A8 on
experimental P aeru~inosa infection in mice (3):-
In the same manner as in Example 6, the survivalrate of mice and the LD50 value were observed for evaluation
of the therapeutic effect of Antibody IN-2A8 at a dose of 1
ug/head against Serotype M strains of P. aeruginosa chal-
20~8~
_ g9 ~
lenge. In this example, however, an immunosuppressant (i.e.cyclophosphamide, Shionogi & Co., Ltd.) was intraperi-
toneally administered to the mice at a dose of 250 mg/kg 4
days before the burning and the bacterial infection so as to
induce the decrease of granulocytes. The results are shown
in Table 7.
.rl -
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8 3 ~
- 51 -
As understood from Table 7, Antibody IN-2A8 was
confirmed to be effective on Serotype M strain of PO
aeru~inosa in the experimental infection on neutropenic
burned mice. Further, ~he range between the upper and lower
limits of the LD50 values with the 95 % confidence limit is
not overlapped with that of the non-mediated group, and the
difference is thus significant. It follows therefrom that
the treatment with the IgM antibody has a distinct
therapeutic effect.
Example 11
Therapeutic effect of Antibody IN-2A8 on
experimental P. aeruginosa infection in mice (4):-
The therapeutic ffect of Antibod~ IN-2A8 on the
associated use with Antibiotic Imipenem l"Thienam"~, Banyu
Pharmaceutical) was examined. In the same manner as in
Example 6, the survival rate of mice and the LD50 value were
observed for evaluation of the therapeutic effect of
Antibody IN-2A8. In this example, however, the mice was
intraveneously administered the antibody as well as the
antibiotic one hour after the bacterial infection. For
suppression of the inactivation in kidney and the renal
toxicity, an eqivalent amount of cyrastin was incorporated
into the antibiotic.-- The results are shown in Table B.
3 8
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u~ O ~ ~ ~ ~r
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_ , _ _ _ _
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2 ~ 3 8
- 54 -
As apparent from the survival rate and the
LD50 values in Table 8, the associated use of Antibody
IN-2A8 and Antibiotic imipenem showed a prominent thera-
peutic effeet in comparison with their sole use. It follows
therefrom that the antibody and the antibiotic work syner-
gistically to exert their therapeutic effects..
Example 12
Influence of Ca++ ion concentration on the binding
property of Antibody IN-2A8:-
Influence of Ca ion concentratin on the bindingproperty of Antibody IN-2A8 was studied by ELISA using 96
well microplates on which the strain IDD1002 of P.
aeruginosa was fixed. The results are shown in Table 9,
from which it is confirmed that the Ca~+ ion concentration
in the buffer solution on the progxess of the antigen-
antibody reaction affords a great influence on the binding
property of the antibody to the flagella of P. aeruginosa.
Table 9o ELISA value ~OD~05~
Ca Concen- ¦ Concentration of Antibody IN_2A8 (~g/ml)
tration _ _ _
~ _ 0-007 ! 0 035 0.18 1 0.88
1 ~ 0.01 0.01 0.03 0.10
lO ~M 0.16 0.47 0.65 0.73
100 ~1 0.43 0.82 0.9~ 1.01
1 mM 0.01 0.16 0.71 0.86
10 mM 0.01 0.01 0.04 O. 6a
As shown in Table 9, the antibody did not exert
any binding property when the Ca** ion concentration was
?d~g~8
- 55 -
less than 1 ~M. It follows therefrom that the presence of
Ca ~ ion is essential for the binding property between
antibody and flagella. Maximum binding activity is seen in
the Ca++ ion concentrai~on of around 100 pM, and the binding
property rather tends to decrease when the concentration is
more than 1 mM.
In the same assay as above but using Mg + ion, nc
binding property was observed in any concentration, from
which it may be understood that, among divalent cations,
only Ca~+ ion is essential for the binding property.
