Vol. 175, No. 1, 1991

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 291-297

February 28, 1991

ESTABLISHMENT

Yuzo

OF

MONOCLONAL

ANTIBODIES

ACIDIC

FIBROBLAST

GROWTH

Ichimori,

Yumiko Kinoshita,

Masaharu

Chemistry

Research Research

Senot,

Laboratories,

Tatsuya Koichl

Division,

Ltd.,

Osaka

HUMAN

FACTOR

Watanabe!,

Kondo

tBiotechnology

and Development Industries

Received January

and

AGAINST

Research

Laboratories,

Takeda Chemical

532, Japan

£i, 1991

SUMMARY: Four kinds of hybridomas secreting monoclonal antibodies (MAbs) against human acidic fibrobtast growth factor (haFGF) were established using recombinant haFGF as an immunogen. The recognition sites of four Mhbs designated AFl-52, 81, ll4 and 1el0 for the haFGF molecule were examined by binding studies with synthetic polypeptides and with amino-terminal truncated forms of haFGF. These experiments suggested that AFI-52, II4, and lCI0 Mhbs recognize epitopes within the ]-5, 44-132 and 6-43 amino acid sequences, respectively. However, the epitope recognized by the AF|-81 MAb could not be determined. The sandwich EIA method constructed w i t h t h e s e MAbs w a s s e n s i t i v e to 1.9 pg/well of haFGF and had no cross-reactivity w i t h h u m a n b a s i c FGF, b o v i n e aFGF o r t h e h s t - ] gene product. Q 1991 ~oad~mioP~s~, z~o

Fibroblast a wide

fibroblasts, angiogenic values:

endothelial activity

(bFCF)

A

in vivo

(1).

form with

(}).

number

like

of

FGFs

proteins,

proteins

share

findings

have

course

cultured FGF

(4),

30-90

% amino

revealed

that

cells.

bFGF

(aFGF)

from

FGF-5

by distinct

malignant

function

FGF-6

homology

behave

tissues

new oneogenes

(6),

sequence

that FGFs might

they possess

They

two forms

with

including also

have

by their

, and a basic one with

are encoded

Recently,

(9),

into

activity

in vitro

genes,

pl

a pl they

(2).

derived

acid

glial

ave separated

and

(3).

int-2

and

5.0-7.0

homology

physiological

reported

myoblasts FGFs

cells

to have mitogenie cells

aFGF

mRNAs

hst-]

are known

and mesoderm-derived

a pl of

Although

and

of normal

been

(FGFs)

cells,

55 % amino acid sequence

synthesize

has

factors

of neuroeetoderm-

an acidic

of 9.6 share

growth

variety

(7) were

with

as growth

but also

transforming

were

which

aFGF

factors

isolated.

under

to

FGFThese

and bFGF.

These

not only

in the

in carcinogenesis. activity

found

encode

Indeed,

it

some conditions

(8,9). The important MAbs

quantification for

against

and a useful

the human

of

diagnosis bFGF

detection

have method

FCFs of

from

human

malignant

already

been

for human

diseases. established

aFGF

291

t~ssues

(haFGF)

and

fluids

EIA

methods

(10,11).

is and

therefore specific

However,

a MAb

have not been established.

0006-291X/91 $1.50 Copyright © 1991 by Academic Press, Inc. All rights of reproduction in any form reserved.

Vol. 175, No. 1, 1991

In against

this

report,

haFgF and

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

we d e s c r i b e the p r e p a r a t i o n and c h a r a c t e r i z a t i o n of MAbs the

c o n s t r u c t i o n of

a

s e n s i t i v e sandwich

EIA method f o r

measurement of i t using these MAbs.

MATERIALS AND METHODS Materials: BALB/cmice were purchased from Shizuoka Animal Center (Hamamatsu, Japan). Human bFGF (hbFGF) and h s t - ] mutein (amino-terminal region 1-27 aa truncated form of h s t - ] ) used in the experiments are described elsewhere (I2,13). Synthetic p o l y p e p t i d e s , haFGF[I-9] (amino-terminal peptide of haFGF,]9 aa) and haFgF[133-140] (carboxyl-termina] peptide of haFGF, ]33-140 aa), were provided by Dr. Wakimasu (Tsukuba Research I n s t i t u t e of Takeda Chemical I n d . , Tsukuba, Japan). Bovine aFGF (baFgF) was obtained from R&D Systems, Inc. (Minneapolis, MN). Preparation of haFgF and i t s truncated forms: haFGFwas p u r i f i e d to apparent homogeneity f r o m recombinant E . c o l i (14). E . c o l i expression systems for Mutein I (amino-terminal l-5 aa truncated form of haFGF) and MuteinH (aminoterminal ]-43 aa t r u n c a t e d form of haFGF) w e r e c o n s t r u c t e d based on the expression system for haFgF (]4). Mutein[ was p u r i f i e d by heparin a f f i n i t y HPLC from the b a c t e r i a l e x t r a c t ( ] 4 ) . MuteinH was s o [ u b i l i z e d from i n c l u s i o n bodies with b M urea and then p u r i f i e d by O-Sepharose column chromatography in the presence of 3 M urea.

Immunization: Eight-week-old female BALB/c mice were immunized 3 times at 3week i n t e r v a l s . T h e y were immunized f i r s t subcutaneously ( s . c . ) with I00 ~g of recombinant haFgF e m u l s i f i e d in Freund's complete adjuvant and the 2nd and 3rd time s . c . with the s a m e dose of recombinant haFgF e m u l s i f i e d in Freund's incomplete adjuvant. Following the t h i r d immunization, they were intravenously infused with ]00 ~g of recombinant haFgF in s a l i n e 3 days p r i o r to s a c r i f i c e . Fusion procedure: Three days a f t e r the f i n a l immunization, the spleen c e l l s from immunized mice were mixed with 8 - a z a g u a n i n e - r e s i s t a n t murine myeloma P3Xb3-Ag.8U] (P3U]) c e l l s at a r a t i o of 5:] and fused by a m o d i f i c a t i o n of the procedure d e s c r i b e d previously (15). The c u l t u r e supernatants from the w e l l s , in which the growth of hybrid c e l l s was observed, w e r e assayed for presence of anti-haFgF antibody by an enzyme linked immunosorbent assay (ELISA) method. ELISA for a n t i b o d i e s to h a F G F : P u r i f i e d recombinant haFGF was d i s s o l v e d in 0.0l M sodium bicarbonate b u f f e r (pH 8.5) at a c o n c e n t r a t i o n of I0 ug/ml, and ]00 ~l of the s o l u t i o n was added to each well of a 96-well m i c r o t i t e r p l a t e . The p l a t e was incubated overnight at 4 ° C , and the f l u i d removed. Residual p r o t e i n binding s i t e s in the wells were blocked by adding Buffer A (phosphate buffered s a l i n e (PBS, pH 7.2) containing 25 % Block Ace (Snow Brand Milk products Co., Japan)) followed by incubation overnight at 4°C. One hundred m i c r o l i t e r s of each hybridoma c u l t u r e supernatant was added to each of the haFGF-coated wells and incubated for 2 hr at 2 5 ° C . After washing the p l a t e 5 times with PBS, 100 ~l of a 5000-fold d i l u t e d s o l u t i o n of h o r s e r a d i s h peroxidase (HRP) conjugated goat anti-mouse IgG (Organon Teknika Corp., West Chester, PA) was added to each well. Following a 2 hr incubation at 25°C, the p l a t e was washed 5 times with PBS and ]00 ~l of p e r o x i d a s e - s u b s t r a t e (22 mg of o -phenylenediamine and 10 ~l of hydrogen peroxide in I0 ml of 0.] M c i t r a t e b u f f e r , pH 5.5) was added. A f t e r 30 min. of r e a c t i o n at 25°C, ]00 ~l of 4 N s u l f u r i c acid was added to the w e l l s , and the absorbance of each well was measured at 4q2 nm using a microplate reader (MTP-32, CORONA Co. L t d . , JAPAN ).

Neutralizing activity of antibody t o haFGF: Mitogenic activity o f haFGF was monitored using BALB/c 3T3 clone A31-1-1 c e l l s as previously d e s c r i b e d (8) and the e f f e c t s of hybridoma supernatant a g a i n s t t h i s a c t i v i t y w e r e i n v e s t i g a t e d . B r i e f l y , 2×103 A3I-I-I c e l l s were suspended in ]60 pl of Dulbecco's modified E a g l e ' s medium supplemented with 0.5 % c a l f serum, then 40 ul of a 5 - f o l d s e r i a l l y d i l u t e d hybridoma supernatant and I0 ~I of a mitogen s o l u t i o n containing 20 pg of haFgF and 10 ~g of heparin were added. After the c e l l s were 292

Vol. 175, No. 1, 1991

BIOCHEMICAL AND BIOPHYSICAL RESEARCH C O M M U N I C A T I O N S

incubated for 18 hr at 37°0, they were pulse-labeled with [3H]-thymidine (37kBq/well) for 5 hr, and subjected to radioactivity determination.

Antibody

production

and

purification:

Each

hybridoma

was

injected

intraperitoneally (i.p.) at 2X10 ~ cells into BALB/c female mice that had received 0.5 ml of mineral oil i.p.. Ascitic fluids were collected 7-10 days after the injections. MAbs were purified by ammonium sulfate precipitation followed by hydroxyapatite HPLC column chromatography. Purified AFI-52 MAb was conjugated with HRP described previously (10). Sandwich EIA for haFGF: Each purified MAb was dissolved in 0.01 M sodium bicarbonate buffer (pH 8.5) at a concentration of 10 ~g/ml. One hundred microliters of each solution or a mixture of the three were added to each well of a q6-well microtiter plate. One hundred microliters of various concentrations of haFGF diluted with Buffer A was added to each well and incubated overnight at 4°C. The plate was then processed for measurement of AFI-52-HRP conjugate binding activity (added from a 200-fold dilution) as described above under "ELISA for antibodies to haFGF". For serum study, the sandwich EIA method was modified as follows. Sixty microliters of the sera or "0" serum containing the standard haFGF and 180 ~i of Buffer B (Buffer A containing 0.3b M NaCI and 13 ~g/ml unrelated mouse IgG) were mixed. The "0" serum was pooled-sera which passed through the AF1-114-coupled Sepharose 4B. One hundred microliters of this mixture were added to each well and incubated overnight at 4°C, then the detection of immuno-reactive haFGF was measured.

RESULTS

Hybridoma s e c r e t i n g a high

antibody

seeded the

antibodies

titer

into 960 wells

growth

to

against

haFGF

in

haFGF:

their

of microculture

of hybridomas

Spleen

sera

were

plates.

was observed

cells

fused

from mice

with

One to 2 weeks

in 390 wells.

P3UI

showing

ceils

and

after the fusion,

Culture

supernatants

were

harvested from each well and assayed for the presence of haFGF antibody using an ELISA method.

Hybridomas

from 4 wells

were

cloned

by limiting dilution

from

BALB/c

mice

their monoclonal studies.

The

as

feeder

ceils.

antibodies

The

subclass

of

that of AFI-II4 and ]CI0 MAbs was 71~

haFGF

were

of

forms

of

114

MAb

by

(haFGF[I-9]

haFGF

(Mutein I

but not

bound

haFGF[133-140] These

results

within

the

recognition

recognition

recognized

polypeptides

haFGF[]-9]

MAb

to

MAbs,

that AFI-52,

44-132

and

6-43

haFGF.

activity

Next, of

haFGF

we to

neutralizing activity

determine

and

BALB/c

selected

MAbs

was

72b~,

1-43

aa

these

(data not shown).

293

to

The AF1-52

and

and

MAbs

can

However,

to ]).

The

it bound only to truncated forms

neutralize of

to

epitopes

respectively.

because

all

not

(Table

ICl0 MAb recognize

sequences,

bound

The AFI-

but

but not to M u t e i n H

the

truncated

MAb

form)

of

synthetic

form).

or to amino-terminal

these

cells.

MAbs

truncated

and and acid

regions

amino-terminal

I-5 aa truncated

to M u t e i n /

amino

which

of

examined.

AF]-I]4

whether 3T3

were

to

MAb could not be determined

examined

to haFGF

1C10) were used for all

and AFI-8]

binding

(amino-terminal

1CI0 MAb bound

site of AFI-8]

To

haFGF[]33-140])

M u t e i n H (amino-terminal

and the

titer

(5×I0 s cells/ml)

.

haFGF but not to the synthetic polypeptides of

AFI-52

site:

four

clones

AF]-114 and

and M u t e i n H ) was

to M u t e i n l

indicate I-5,

the and

of thymocytes

representative

(AF]-52, AF]-81,

immunoglobulin

Determination

that showed high antibody

in the presence

these

the mitogenie MAbs

had

no

Vol. 175, No. 1, 1991

BIOCHEMICAL A N D BIOPHYSICAL RESEARCH COMMUNICATIONS

Table

1.

Binding

ability

o f MAb t o v a r i o u s Binding

MAb

haFGF

AFl-52

+~'

AF1-81

+

AF]-II4

+

1010

+

antigens

ability"

Pepl-9

Pep133-140

+

-'

-

-

-

-

+

-

+

-

MuteinI

Mutein

+

One hundred microliters of hybridoma supernatant was added to each well coated with antigen: (Pepl-9: amino-terminal peptide of haFGF, ]-9 aa ; P e p 1 3 3 - 1 4 0 : carboxyl-terminal peptide of haFGF, 133-140 aa ; Hutein I : amino-terminal ]-5 aa truncated form of haFGF; H u t e i n M : aminoterminal 1-43 aa truncated form of haFGF.). The binding of MAb to the well was examined with goat anti-mouse IgG antibody labeled with HRP. h Bound. Not bound.

Construction

of

quantified

by a

them and

(AF]-52 ]C10)

EIA

sandwich

HAb)

or

plates.

from

coated

wells

].5 p g / w e l l

was

the

microtiter

was

method

haFGF:

To

examine

whether

EIA,

four

kinds

of HAbs

were

labeled

with

HRP.

Each

the o t h e r

mixture As with

for

of

shown

all in

three

Fig. I~

the m i x t u r e .

was

the

The

of

used

most

purified

to

coat

limit

could

be

and used.

One of

(AF]-8],

AF]-]]4

HAbs

sensitive

detection

haFGF

wells

result

using

of was

this

96-well obtained

EIA

method

of haFGF.

1.O 1.0 04 04 CrJ

v

v ¢D

~_ JQ o c9

Establishment of monoclonal antibodies against human acidic fibroblast growth factor.

Four kinds of hybridomas secreting monoclonal antibodies (MAbs) against human acidic fibroblast growth factor (haFGF) were established using recombina...
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