Vol. 88, No. 2, 1979

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATlONS

May 28, 1979

Pages 575482

MONOCLONAL ANTIBODIES AGAINST PURIFIED NICOTINIC ACETYLCHOLINE RECEPTOR Christopher

M. Gomez',

David

Barry 1 Department

1* , Phillip

P. Richman

G.W. Arnasonl

W. Berman',

and Frank

2 Neurobiology Received

Laboratory, March

Salk

Institute,

A. Burres',

W. Fitch3

of Neurology and 3Department of Pathology, Sciences and the Pritzker School of Medicine, Chicago, Illinois 60637

Biological of Chicago,

Steven

San Diego,

Divisitm of the The University

California

92112

12,1979 SUMMARY

Eleven stable monoclonal hybridoma cell lines synthesizing antibodies against purified acetylcholine receptor from Torpedo californica were Spleen cells from a rat immunized with acetylcholine receptor produced. and exhibiting experimental autoimmune myasthenia gravis were fused with a mouse myeloma cell line: Studies of the binding of these antibodies to acetylcholine receptor by use of a passive hemagglutination assay in the presence of various cholinergic ligands revealed four general categories of binding specificities: 1) blockade only by alpha bungarotoxin, 2) partial blockade by all ligands, 3) increased titer .in the‘presence of alpha bungarotoxin and benzoquinonium chloride, 4) absence of effect by any ligand. INTRODUCTION Anti-sera useful

tools

zation

of these

of multiple

in

populations

a means by which be obtained. have

applied

ceptor (AChR). * To whom reprint

this

reagents

Recently

this

protein

should

developed

in this

of nicotinic has been

techniques

provided

and specificity manner

of macromolecular

to the study

to be

irmnunochemical

(2) have

structure

prepared

probes

in their

(mAbs)+

of uniform

to be powerful

requests

differ

antibodies

immunoglobulin

Although

which

have proven

and --in situ localigeneral, are made up

in

specificities.

of monoclonal

approach

antigens

Such anti-sera,

of antibodies

Immunochemical prove

purified

quantification,

(1).

and antigenic

the production

doubtedly

against

the identification, antigens

characteristics for

raised

will

may un-

structures. acetylcholine

intensively

studied

We reby

be addressed

t Abbreviations: mAb, monoclonal antibody: AChR, nicotinic acetylcholine receptor; EAMG, experimental autoimmune myasthenia gravis; aBT, alpha bungarotoxin; Cm, carbamylcholine; dtc, d-tubocurarine; atr, atropine sulfate; Bz, benzoquinonium chloride; Brij-PBS, phosphate buffered saline with 0.05% Brij 35; RBCs, sheeperythrocytes; HA, passive hemagglutination assay. 0006-291X/79/100575-08$01.00/0 575

Copyright All rights

@ 1979 by Academic Press, fnc. of reproduction in anyform reserved.

BIOCHEMICAL

Vol. 88, No. 2, 1979

biochemists

and biophysicists,

lationship

between

the subunit range

its

4 distinct

can be altered

through

gravis

develop

(EAMG),

Monoclonal ceptor

cule spleen

the model

cells a myeloma

lines

producing

of the binding

for

for

disease,

with

valuable

tools

identifying

regions agents,

the induction

from

rats

cell

of these

known to modulate

with

to produce

anti-receptor

ligands,

antibodies receptor

snake

neurotoxins, purified

autoimmune

Tor-

myasthenia antibodies

(6).

on the

surface

of re-

elucidating

the

subunit

structure

of the molecule defining

We report

to AChR inthepresence

for

on the mole-

described

AChR and exhibiting of stable

important sites

In the studies

a number

antibodies.

of which

with

of anti-AChR

to

areas

and for

of EAMG.

immunized

line

for

thought

the properties

experimental defined

Even

structures is

immunized

the appearance with

protein

(5)

animals

reactive

of pharmacologic

with

stances

In addition,

provide

crucial

states

the re-

unanswered.

proposed

This

the use of cholinergic

concomitant

protein

the binding

(3,4).

concerning

remain

unresolved,and

functional

antibodies

should

of this

of AChR is

anesthetics.

pedo AChR

questions

and function

1 and 4 polypeptides

have at least and local

fundamental

structure

composition

between

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

below,

EAMG were hybridoma

preliminary

fused

cell studies

of various

sub-

function. METHODS

Pharmacologic Agents - Alpha bungarotoxin (aBT), [lz51]aBT, and alpha neurotoxin were kindly supplied by Dr. James Patrick. Carbamylcholine (Cm), d-tubocurarine (dtc), atropine sulfate (atr), choline bromide and physostigmine were obtained from Sigma (St. Louis, MO), neostigmine methylsulfate from Elkins-Simms (Cherry Hill, NJ) and sodium acetate from Fisher (Fair Lawn, NJ). Benzoquinonium chloride (Bz) was a gift of Sterling Drug Company (Rensselaer, NY). All solutions were prepared daily. AChR Preparation - AChR was prepared from the electroplax of Torpedo californica by the method of Berman and Patrick(7,g). Approximately equal proportions of monomeric and dimeric AChR (3.8-4.0 nmoles of aBT binding sites per mg protein) were suspended in phosphate buffered saline containing 0.05% of the non-ionic detergent Brij 35 (Cal Biochem, San Diego, CA) (Brij-PBS). Immunization - AChR in 0.05% Brij-PBS was emulsified with an equal volume of complete Freund's adjuvant and injected in multiple intradermal sites along the back and in the footpads of 200 gm female Lewis rats. In the primary immunization each animal received 45ug of AChR protein. At the same time each animal was injected with forty-five microliters of killed B.pertussis vaccine (gift of Eli Lilly Co., Indianapolis, IN) at multiple intradermal sites. All animals developed the typical clinical course of EAMG(9) with frank weakness beginning on day 9-12 after inoculation and clearing by day 21. On the 24th day, at which time the acute phase of the disease had cleared but prior to development of the chronic phase, animals received a second injection of AChR, 25 to 1OOug without adjuvant, intraperitoneally. Spleen Cell Hybridization (10) - Three days after the second injection of AChR, spleens were removed from immunized animals, homogenized in a ground glass homogenizer, and centrifuged over a Ficoll-Hypaque gradient (S.G.: 1.094). The mononuclear cell suspension thus obtained was then fused with

576

Vol. 88, No. 2, 1979

BIOCHEMICAL

AND BfOPHYSICAL RESEARCH COMMUNICATIONS

the mouse myeloma cell line P3-X63-Ag8 (kindly provided by Dr. C. Milstein). Spleen cells (5 x 107) and myeloma cells (5 x 106) were mixed in 6Omm Petri dishes (Falcon) and centrifuged at 250xg for 3 minutes. The cells were then flooded with 50% polyethylene glycol (PEG 1500, Fisher, Fair Lawn, NJ), cells were cultured overnight in Dulbecco's modified and after washing, Eagles medium with 20% heat-inactivated fetal calf serum. The cells were then cultured in hypoxanthine-aminopterin-thymidine (HAT) medium in microtest wells (Costar, Cambridge, MA) (approximately 1 x lo4 cells per well). Supernatants from wells with proliferating hybrids were assayed (see below) for anti-AChR activity. Cells from positive wells were then cloned by limiting dilution in microtest wells with irradiated Lewis rat thymocytes serving as a feeder layer. Clones were grown up in bulk in spinner flasks. Some clones were re-cloned by limiting dilution. Antibody Assay - A passive hemagglutination assay (HA) was performed by modification of the method of Aarli et al. (11). AChR (T californica) in 0.05% Brij-PBS was coated onto sheep erythrocytes (RBCs) $eviously treated with 83.3ugfml tannic acid. The cells were then washed and resuspended in 0.05% Brij-PBS. The ligand-binding properties of the RBC-bound AChR were tested in a competitive binding assay. AChR-coated RBCs were incubated with various ligands (10B5M) in 0.05% Brij-PBS for 30 minutes prior to a 30-minute incubation with [1251]aBT (10e7M). The cells were centrifuged through a layer of n-butyl phthalate and the pellets counted in a gamma counter. Serum or culture supernatants were serially diluted with 0.05% Brij-PBS and 1% non-immune heat-inactivated rabbit serum. The AChR-coated sheep erythrocytes were then added to the serum (or supernatant) dilutions in round bottom microwell plates (Falcon, Oxnard, CA), incubated at 3J°C for 15 min., and scored for agglutination visually and also after centrifugation at 250xg for 1 min. (12). Assessments of blockade of antibody binding in the HA were carried out by preincubation of AChR-coated cells with pharmacologic a ents in 0.05% Brij-PBS or with 0.05% Brij-PBS alone B to their addition to the serum or supernatant for 30 minutes at 20 C prior dilutions. RESULTS Hybridization:

All

of spleen 217 .

cells

Half

from

were

layer

wells

grew within number that

that

(Bk) whose

serum HA titer

out hybrids.

Seventy

for

the well

as a single

antibody-producing

(see below) more than

wells dilution

were

with

from which from

the

they

were

same well

are

were

positive were

re-cloned for

antibody

monoclonal.

of the mAbs remained

lo%-30%

Clones

stable

to AChR was

percent

anti-AChR

hybridomas. patch.

clones

clones

contained

proliferating

well

derived (n=25)

the hybridization

rat

by limiting

the original

from

grew

hybrid-positive

of the primary

resulted

a single

positive

subclones

here

planted

obtained

some clones

these All

from

the wells

supernatants Clones

mAbs studied

of planted

All were

of the

activity. feeder-

such hybridomas

designated

derived.

by the

(It

is

possible

identical.)

Five

by limiting

dilution.

production

The binding

of

suggesting specificities

as the hybrids

were

carried

through

10 passages.

mAb Specificities:

The HA titers

of culture

receptor

are

in Table

hybridomas

presented

577

supernatants 1.

Most

from

supernatants

11 antiwere

Vol. 88, No. 2, 1979

8lOCHEMlCAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Table 1 Anti-AChR BA Titers (Log2) of Various Cholinergic Ligands * Ligand BZ aBT dtc GUI

Supernatant in the Presence Clone Control

atr

60D

13

0

13

12

12

13

561 56K 57L 625

29 22 16 23

24 20 14 18

24 21 14 18

21 22 14 20

24 20 14 20

21 21 14 18

73G

17

20

17

21

17

17

381 58E 58F 77A 77F

13 9 6 19 20

13 9 6 19 19

13 9 6 19 20

13 9 6 19 20

13 10 6 20 20

14 9 6 19 20

i

dtc,

assayed

Bz, Cm, atr - 10e514; aBT - 10m7M

at least

in which

3 times,

5 replicates

95% confidence clones

obtained

from

are not titers

RBCs in Brij-PBS

supernatants.

representative

carried

of these

cultures

Control coated

were

limits

between

with

out

titers

significant

since different

alone

Each supernatant

Differences

the supernatants cell

to the addition

was also

tested

the

in HA titers tested

were

densities.

of preincubation

of the AChRof antibody-containing

against

uncoated

RBCs was

detected.

The pharmacologic activity of RX-bound AChR was tested by radioassay (see Methods). Binding of ['251]aBT to AChR-

RBCs was inhibited

of effectiveness:

no agglutinating

activity

RBCs treated

coated

Brij-PBS;

Experiments

and tanned a competitive

with

the result prior

given.

on the same day determined to be +2l.

containing are

results

by cholinergic

ligands

in the

following

order

aBT>dtc>Bz>Cm>atr.

The effects of various cholinergic ligands on antibody binding in the HA are indicated in Table 1. In control experiments each ligand was tested for, and found to lack, agglutinating activity against the AChR-coated acetate,

RBCs. choline

Preincubation of the receptor-coated bromide, neostigmine methylsulfate,

578

RBCs with

sodium

and physostigmine

Vol. 88, No. 2, 1979

(all

BIOCHEMICAL

AND EIOPHYSICAL RESEARCH COMMUNICATIONS

10V5M)

had no effect on the HA titers of any of the hybridoma superThe data allow roughly four categories of mAb specificities In the first category, represented by hybridoma 60D, to be distinguished.

natants.

hemagglutination

was completely

ligands.

The second

57L,

was that

625)

and by all

other

antagonist,

type

creased

it.

from with

unaffected

The inhibitory

effect

60D was studied

half

The possibility following

that

from

reacted

Resuspension natant

with

the possibility against

that

30 minutes

natant

with

resulted

Free

aBT thus

cating

that

between

with

and addition

with

from

does not

affect

the antibody

AChR-coated

a competitive

equal is

for

of 625 superexperiment

actually

be directed

RBCs were

preincubated

inhibitor values

hemagglutination

10e7M c&I

agglutination.

of aBT binding.

30 minutes)

to control

directed

with

dilution

60D could

degrades

out by the

In another

clone

1)

6 x 10WvM and 8 x lo-'M.

no resultant

of a 1:27

aBT (10V7M

in HA titer

(Figure

preincubated

hemagglutination.

10V4M dtc,

incubation

the concen-

curve

of the RBCs was ruled

aBT and not AChR was tested.

Subsequent

by antibody

Increasing

60D supernatant

antibody

and Bz in-

tested.

inhibition

RBCs were

in definite

in which

381, 58E and F, and 77A and F

ligands

AChR-coated

of the cells

resulted

that

of aBT and 60D supernatant

the surface

by aBT

the muscarinic

however,aBT

detail.

the combination

it

blocked

of CYBT on hemagglutination a steep

by other

561 and K,

73G) is

hybridomas the

at aBT concentration

experiment.

and then

includes

in greater

effect

including

(hybridoma

by all

effect

(hybridomas

the HA titer;

the HA produced

maximal

AChR or releases

for

category

were

of aBT in

tested,

reduced

little

was partially

specificity

tested

The final

clone

tration

ligands

The third

HA titers

observed

hemagglutination

cholinergic

atr.

by aBT with

of specificity

in which

none of the ligands where

blocked

and then for

60D super-

60D (i.e.,

213).

by 60D supernatant,

against

AChR and not

indi-

against

the

toxin.

DISCUSSION The experiments tion

of the binding

purified bodies

nicotinic were

AChR could binding bodies dtc

which

in

several

cholinergic

of monoclonal

receptor.

studied,

and the binding

of six

by cholinergic

ligands.

could

completely competitively ways. ligands

inhibit tested

Four

aBT.

all

is bind

but

In the first

This that to the

579

not result

against

by other

of mAb type

of

RBCs by antiligands

such as

may be accounted

antibody same site

anti-

to erythrocyte-bound categories

of receptor-coated

by aBT,

One possibility

investigaprepared

hybridoma-derived

of these

be distinguished.

60D) agglutination inhibited

preliminary antibodies

Eleven

be altered (hybridoma

is

above represent

(13)

acetylcholine

specificities

specificity

presented

properties

60D, uBT, on AChR.

for

and the QBT is

BIOCHEMICAL

Vol. 88, No. 2, 1979

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

2’0 2e-

? l-

26-

24-

a Bungarotoxin

Figure

to bind

to AChR in an essentially

other

ligands

studied

much less be similar

than

then

60D antibody

account

is

site.

from

possibility the

ligand

binding, the

site, would

binding

results

60D binding

site

73G are difficult sites

that

than

antibody explanation

in close

and would

proximity

be expected

Steric

hindrance

at locations would

60D binds

oBT binding,

60D which

any of the other

ligands

change

by

and 10e7M cd3T does

binding

antibody

would

hemagglutination

smaller

they

facilitation

towards are

Further

by ligands by antibodies

to interpret.

directed

or that

conformation.

predicted

antibody

and that

inhibition

of hemagglutination

either

Cm,

dtc,

ligands to

by

peripheral

not be effective.

at a site but not

in AChR which

remote

other

ligand

occludes

the

site.

and 625 and the apparent are

be that

in a conformational

The partial tested)

while

with

dtc

of the AChR surface.

to inhibit

binding

60D binds

60D binds

8,000)

the

than

and suggests An alternative

antibody

approximately

portion

be expected

that

while

antibody

that

aBT is much larger

after

affinity behavior

of lo-%

for

mask a considerable

A third

binding

in the presence

weight

If

The observation

the data

fashion,

much greater

interpretation binding site.

(molecular

aBT might

reversible. with

support this to the ligand binding

to the ligand

irreversible

of antibody

observed.

can occur

to the ligand

readily caBT but

the type

to that

not, however, does not bind

tested

are

affinity

and atr,

could

(Ml

1 - Anti-AChR HA titers of hybridoma clone 60D supernatant AChR-coated FzBC preincubation with various concentrations of CXBT

known

Bz,

Concentration

(at

the single

from hybridomas of hemagglutination

concentration 561 and K, 57L by hybridoma

These results suggest that the antibodies antigenic determinants close to ligand binding

capable

of detecting

investigations

studying

580

ligand-induced a wide

changes range

of ligand

in AChR con-

Vol. 88, No. 2, 1979

centrations, assay

and making

(7,8)

will

A somewhat tion

antagonist

observation

We know of no prior binds

to Torpedo

to compete

with

remote

with

very

differences

from high

characteristics blocking

assays than

identified

investigations

using

of cholinergic

from

tested.

sites,

or,

it

derive with

(15).

may bind they

from

may

structural

active

respect

(Preliminary

381,

Some of the

are particularly

differently

re-

AChR (14).

These

site.

is

hybridomas

conversely,

binding

in

to ligand

results

have

and 77F as IgM.)

of the monoclonal

antibodies

the nicotinic reagents

of AChR, ofthe

muscarinic

to Electrophorus

antibodies

IgM antibodies behave

of hemagglutina-

this

concentration

in the HA may also

as IgG-2a

these

that

ligands

ligand

possibilities.

blockade

binding

immunoglobulins

by which

these

at high

of the

binding

and might

60D and 625

and pharmacology

for

observed

do other

new tools

suggesting

to the

The availability provides

the partial

by the

the ligand affinity

of the mAbs.

agglutination

is

titers

such as radioimmuno-

between

AChR, however,

unaffected

in behavior

assays

data

a-neurotoxin

P, and 77A and F are to sites

antibody

to distinquish

The hemagglutination

bind

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

use of other

be required

surprising

by atr.

ported

8lOCHEMlCAL

described

AChR may be explored.

will

include

pathogenesis

studies

of the

above Future structure

of EAMG, and of the mechanisms

transmission. ACKNOWLEDGMENTS

We thank stages This

of this work

4197)

Dr.

project

was supported

and the Muscular

post-doctoral Fellowships

Fellowship from

Tom McKearn

for

his

and Susan Tonsor in part Dystrophy

extensive for

by grants the NIH

the Myasthenia

Gravis

excellent from

Association

from

help

technical

the NIH (NS13526,

of America,

(NSOS715) Foundation,

in the early

as well

and H.R. Viets

assistance. A19268, as a Student

Inc.

REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11.

Yalow, R.S. (1978) Science =,1236-1245. Kohler, G. and Milstein, C. (1975) Nature =,495-497. Heidmann, T. and,Changeaux, J.P. (1978) Ann. Rev. Biochem, 67,317-357. Raftery, M.A., Vandlen, R.L., Reed, K.L., and Lee, T. (1976) Cold Spring Harbor Symp. Quant. Biol. x,193-202. Steinbach, J.H. (1979) Cell Surface Reviews, Poste, G., Nicolson, G. Publishing, Amsterdam, in press. and Cotman, C. eds., North Holland Patrick, J. and Lindstrom, J. (1973) Science =,871-872. Patrick, J.,Lindstrom, J., Culp, B., and McMillan, J.'(1973) Proc. Nat. Acad. Sci. USA z,3334-3338. J. Submitted for publication Berman, P.W. and Patrick, Lennon, V.A., Lindstrom, J. and Seybold, ME. (1975) J. Exp. Med. x,1365-1375. McKearn, T.J., Sarmiento, M., Weiss, A., Stuart, F.P. and Fitch, F.W. (1978) Current Topics in Microbial. Immunol. &,61-65. E. (1975) Stand. J. Immunol. Aarli, J.A., Mattsson, C. and Heilbronn, &,849-852.

581

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BIOCHEMKAL

AND BIOPHYSICAL RESEARCH COMMUNKATIONS

Severson, C.D. and Thompson, J.S. (1966) J. Innuunol. 95,785-789. Gomez, C.M., Richman, D.P., Berman, P.W., Burres, S.A., Arnason, B.G.W. and Fitch, F.W. (1979) Fed. Proc. (in press). Lindstrom, J. and Patrick, J. (1974) Synaptic Transmission and Neuronal Interaction, M.V.L. Bennett, ed., pp. 191-216, Raven Press, New York. Greenbury, C.L., Moore, D.H., and Nunn, L.A.C. (1963) Immunology &421-428.

582