Note: Descriptions are shown in the official language in which they were submitted.
4 c t . 1 ~ 5 13 ~ ~ ~ G G s ~ ~ e r i e ~ ~; 1 9--5 ~ ~i-- 1 ~ 8 0 ~ . 3
,, , ~ ,-,~, ".
~ONOCLONAL ANTIBODIES AGAINST R~CEPTOR-
INDUCED BINDING SI~ES
~e~c~ip~lQn
~eahnlcal ~ield
~ he present inven~ion rela~es ~o an antibody
binding ~ite that iG induced on a ligand when that ligand
is bound by a receptor to fo~m a recep~or-lig~nd complex.
Monoclonal anti~odie~ that ~mmunoreact with the receptor-
induced binding ~ite and therapeutic and diagnosticmethods ucing the monoclonal an~ibodies are al~o
cont~mplated.
Baakground
Interactions of ligar,d~ with cell ~ur~ace receptor
mole4ule~ are hallmark~ of biological processes.
Exemplary of such interactions are the binding of the
ligan~ formed by a portion o~ the envelope protein
(gpl40) of the AIDS ~irue (H~V) with the CD4 raceptor on
T ~ells, the receptor6 of t~e ma~or hi~tocompatibility
complex that interact with numerous ligands in the
processes of self~non-self di~crimination in the
immunological sy~tem, and t1s interaction of the
fibrinogen li~and with the ~PIIb/IIIa cellular receptor
on platelet~. The ~ibrinogen:GPIIb/IIIa ligand~receptor
pair are of particular intere~t in blood olotting,
thromb~s formation, and are utilized hsreinafter as
exemplary of ligandR and rsceptors.
The art ha~ long ~ought immunological methods to
distinguish the bound and non-bound form6 of ligands and
recep~or-~ because that ability would allow ~or a
datermination of the state o~ the phy~iolo~ical
mechanisms mediated ~y receptor: ligand complex
formation. Until recently, efforts to make ~uch
determination5 by immunological met~ods have been
Oc t . 1 ' F~ 3~ ~ ~3 ~13 G~iSSacM se r i ec S 15~--54~ - 13~: ~ P . 4
Z~ 4
- 2 -
frustrated becau~R biological ~amples ~an contain ligands
and receptor~ in both ~ound (complexed) and free forms
For instance, the vas~ulature of individ~als
undergoing a thrombotic event contains non-~ound form3 of
flbrino~en and GPIIb/IIIa as well as platelets having a
fibrinogen GPIIb~IIIa complex on their surface The
fibrinogen GpIIb/IIIa complex expresses antigeni~
determinants common to non-bound fibrinogen and non-bound
GPIIb/IIIa, thus making it difficult to identify a
thrombotic condition or thrombus location by existing
immunological metho~
Re~ently, Frelinger et ~1., J Biol Chem ,
263 12397-12402 (1988) reported identifying an anti~enic
determinant expre~sed by GPIIb/IIIa only when ~PIIb/IIIa
was bound to a ligand That i8 ~ the antigenic
determinant described by Frelinger et al was expre~ed
by the recep~or of ~he reCeptor ligand complex
Others have reported tho preparation of monoclonal
antibodie~ that immunoreact with an antigenic de~e~mi~ant
expressed upon liga~d binding to artificial, non-receptor
surface~
Soria et al , ~ _5ællgu~___terface Sci , 107 204-
208 (1985) de~cribe a particular monoclonal antibody
referred to as DS~a2 that wa~ induced by immunization with
cross-linked fibrin fragment and ~inds to pl~stic-
adsorbed ~ibrinogen and al50 to the so-CallQd fibrinogen
D fragment adsorbed on pla~tic or free in solution, but
doe~ not bind ~o solution pha6e f~brinogen or the so-
called fibrinogen E ~ragment when in solution
Nilsson et Al , M~lec Immunul , 24 48~-g4, 19~7,
~escribe the preparation of monoclonal antibodies that
immunoreact with complement- C3 fragments when particle
bound to Zymo~an A, ~ut not with soluble C3 Pra~ments
The natur- of protein binding to Aymosan A involves
covalent ohemloal bond~
Oc t . 1 ' ~5~ 37 ~ 1 DGSS~c~ se r i es <S 1~--5~ 13~: 0 P . 5
2~)00~4
- 3 -
In another report Abram~ et al., ~lood (Sup~l, 1~,
70:355a (Dec. 19~7) briefly di6cuss a monoclonal antibody
designated 9F9 that is said in the published ab~tract to
bind to platelet-bound fibr~.nogen. That brief
disclosure, however, did no~ characterize the specific
hinding propertie~ of monoclonal 9F9.
Numerou~ monoclonal antibodies that immunoreact
with solubilized ~ibrinogen have been reported. One ~uch
report i~ Lindo~ et al., Blood, 68:355-362 (Aug. 1~8~).
~hat paper r~ported thQ use of commercially available
anti-human fibrino~en monocl.~nal antibodieS as well as
afflnity-puri~ied polyclonal an~ibodies to the D and E
fragments.
It ha~ now ~een ~ound that ligands that are
~pecifically bound to a re~ep~or ~an be distinguished
fro~ non-bound ligand~ by th~ pre~ence of a receptor-
induced antibody binding æite ~RIBS) expressed b~ the
receptor-bound ligand. That is, a cla~e of antigenic
determinants ha~ been discovered which are expressed when
a ligand 6pecifically binds to a re~eptor but are not
express~d by the non-bound r~ceptor or the non-bound
ligand.
A RIBS is ~xpr~ssed on a liyand due to the
~pe~i~fia inte~aotion o~ a ligand binding to its cognate
receptor. A RIBS i~ not an antigenic determinent ~ite
that is exposed when a prot~in interact6 non-~peai~ically
with another 6urface, fiuch a~ pla~tic-absorbed pro~ein,
or when a protein-interacts by covalent chemiaal bonds
with a ~urface. Theoe latter two ~xamples are di6closed
in the Soria et al., and Nilsson et a}., re~erences
described above, and do not involve ~pecific receptor
ligand ~inding intera~tion that produce a RIBS as
disclo~e~ herein.
0-= t . 1 ~ 3~ el 13 013 GGSS~c~l se r i ~5 ~ 1~--5~6-- 131~: el P . ~S
)044
- 4 -
The pre~ent invention relates to antibody
molecules that lmmun~rea~t k~ith a ligand, preferably
fibrinogen, when it is bound ~o a receptor, but do no
immunor~act with ~oluble ~ibrinogen, as for example in
plasma.
These antibodies recognize the RI~S induced a~ a
~onsequen~e of the fibrinogen interaction of proteins,
prefera~ly GPIIb/IIIa, on platalet~. The antibodies of
this inven~ion a~t seleatively with bound-fibrinogen even
1~ with a large exce~s of plasm.~ fibrinogen. The unique
proper ie8 of ~hese antibod:.~s therefore allow for a wide
~ariety of diagno~tic ~y~em~ and th~rapeutic methods of
use.
one em~o~iment o~ the pr~en~ invention there~ore
lS contemplate~ a monoclonal antibody that immunoreact~ with
a receptor-induced ~inding site expressed by a receptor
ligand complex. Preferably the ~onoclonal antibody is
selected from the group oon~i~ting o~ 2G5, 2F10, 3Gll,
and 4G~o.
The monoclonal antibody i~ produced by a
hybridoma selected from the group consisting o~ hybridoma
2G5, hybridoma 2F10, hybridoma 3Gl.~, and hybridoma 4G10.
Another embodiment o~ this invention i~ a cell
culture composition which compri~es:
2S a) a hybridoma that p~oduce~ an antibody that
immunoreacts with a r~c~ptor-induced binding ~ite
expre~ed ~y a GPIIb/IIIa:~-brinogen ~mplex, the
hybridoma ~elected from the group con~ ing of hybridoma
2G5, hybridoma 2F10, hybridoma 3Gll and hybridoma 4G10;
b~ a~tibody molecule~ ~ecreted by the
hybridoma: and
c) a culture medium for the hybridom~.
~urther contemplated is a method o~ detecting
the pre~ence of a recept4r-ligand complex wherein the
syste~ comprises ~ monoclonal antibody that immunoreact~
Oc~ t . 1 ~ 38 ~13~ D~:S~c~1 s~r i.es ~ 5~<S-- 1380 p . 7
2000044
-- 5 --
with a receptor-induced binding site expressed by a
receptor-ligand complex, ~ut i6 not expreæ~ed ~y the non-
bound receptor or th~ non-bound li~and. Pref2rably the
receptor complex i~ GPIIb/IIIa:fi~rinogen. Also
5 prefe~red ~ 8 the method wherein ~he monoclonal antibody
ls linked to an in vivo indicating mean~.
Another aspect o~ this invention is the diagnostic
~ystem, in Xit for~, for the as~a~ of a va~cular fluid
sampl~ or the presence o~ the GpIIb/IIIa:fibrinogen
omplex Which ~y~te~ comprlse~ a monocl~nal antibo~y that
immunoreacts with a RI~S expre~sed by this complex.
Another embodiment of thi~ invention is the method
of di~peræement of a thrombus, in vivo, in a mammal which
comprises:
a) intravenously administ~ring to said mammal dn
effectlve amount of a monoclonal antibody-pla~minogen
activatin~ enzyme conjugate wherein the monoclonal
antibady of the a~n~ugate immunoreact~ with a receptor-
induced binding site expressed by a GPIIb/IIIa:fibrinogen
~o complex.
Preferably the plasminogen activating enzyme is
ti~æue plasminogen a4t~vator and the monoclonal antibody
i5 ~elected from the group consisting of 2G5, 2FlO, 3Gll,
and 4Glo.
2S AnoSher use for the i~ vivo diagnostic and
therapeutic abilitie6 of the antibodies of this invention
is the method to detect, and locate a thrombu~ in a
ma~mal which compromi~es the 8tep8 of:
a) intravenou~ly administering to a mammal, an
eff~ctiv~ amount of a monoclonal anti~ody that
immunoreaat8 with a receptor-induced ~inding site
expressed by a CPIIb/IIIa:~ibrinogen complex;
b) maln~aining the adminiæ~ered mammal for a
predetermined time period ~u~ficient ~or the antibody to
immunoreact with the complex and ~orm an immunoreaction
0~ t . 1 ' ~ 35~ DCSSa~r`1 se r i e~ ~ 19--5~-- 1:~:~13 P . a
Z~)00~44
- 6 -
product~ and
c) aæ~aying for the presence of any
immunorea~tion produ~t formed in step b and thereby the
presence o~ the thrombus.
Preferably the antibody i~ linked to an in viv~
indlo~ing ~ean~.
De~ai~ed Desc~iption of t~e Inyention
A. Definitions
~ept~r: Receptor and receptor protein are
term~ used herein to indicate a biologicallY active
pro~einaceous ~olecule that ~pecifiaally binds to (or
with) other molecules, referred to as ligand~, to form a
receptor-ligand protein complex.
1igan~ and Cogn~te ~iga~: Ligand ref~r~ to a
lS molecule that contains a ~tructural portion that i8 bound
by specific interaction with a particu~ar receptor
protein.
~ : A RI BS i s a
neo-antigenic determinant that is expressed ~y the ligand
portion of a receptor-ligand oomplex but i9 not expressed
~y ei~e~ the non-bound ligand or the non-occupied
receptor. A RIBS can be either "conformational" or
"~equ~ntial". A RrBS iB the result of specific
alterations of the ligand induced by receptor binding,
i.e., a "cryptic antigenic determinant".
~ : Re~erY to a neo-
antigenic determinant formed by changes in conformation
of a ligand protein upon binding to its cognate
~speclfic) receptor. Thuæ, a ligand descri~ed herein
does not exprecs a cryptic ~ntigenic determinant unless
the ligand has speaifically bound to a receptor~
"Unit doQe" as it pertains to the inocula of the
present inven~ion refers to phy~ically di~crete units
æuitable a~ unitary dosageQ for animals, each unit
containing a predetermlned quantity o~ active material
O c t . 1 ' æ s ~: 3 5 e~ D ~i ~i S~ l s e r i ~ = c~ 5 ~ ~-- l 3 s e el P . 9
Z00()044
calcul~ted to produce the de~:Lre~ immunogenic effect in
association with the required diluent; i.e~, carrier, or
vehicle The specifications for the novel unit dose of
thi~ inventi~n are dictated ~y and are directly dependent
on (a) the unique characteristics of the active materi~l
and the particular immunologic e~fec~ to ~e achieved, and
(b) the limltations inherent in the art of compounding
su~h active materia~ ~or immunologic use in animal~, as
disalosed in detail herein, the~e being features of the
present invention.
Unit dosage form~ are typically prepared from the
frozen ~r dried antibody by di~per~ement in ~
physiologically tolerable (acceptable) diluent or vehicle
such as water, 6altne ox pho~phate-buffered saline ~o
form an aqueou~ oompo~ition. Such dlluents are well
known in the art and are di~cussed, for example, in
Reming o~n'6 Pharmace~ticaL_~çi~n9~, 16th Ed., Mack
Publi~hing ~ompany, Ea6ton, PA (1~80) at page~ 14~5-14~7.
Do6a~e ~orm~ can al~o include an adjuvant as p~rt
of the diluent. Adjuvants 6uch a~ complete Fr~und's
adjuvant (~A), inaomplete Freund's adjuVant (IFA) and
alum are material~ well known in the art, and are
availabls commercially from several sources.
~ : Antigenic
determinant or antigen re~ers to the actual ~tructural
portion of thR antigen that i8 immunologically ~ound by
an ahtibody comblning site. The terms are al~o u~ed
intar~hang~ably with epitope.
Neo-anti~n: A neo-antigen, as defined herein, is
a new antigenic determinant that was not expressed by a
ligand prior to binding to the receptor but that is
expres6ed in ~he ligand-receptor complex.
Antibodv: -The term an~ibody tn it~ variou~
grammatical forms is u6ed herein to refer to
$mmunoglobulin molecules and immunologicallY active
0-= t . 1 ~ 5~ 13 0 e~ ~ L:~ G S ~ 5~ r i e ~; cS 19--~5~ ~-- 13~ ~ P . 1 el
Z~ X)44
-- 8 --
portions o~ immunoglobulin molec~le~, i.e., molecules
that contain an antibody combining site or paratope.
Exemplary antibody molecules are intact immunoglobulin
molecule~, ~u~tantially intact imm~noglobulin molecules
and portion~ o~ an immunoglobulin moleoule, including
tho~e portion~ ~nown in the art a~ Fab, Fab', F~ab')z and
F(v).
~ ¢~ y~Combininq ~ite: An antibody combining
~ite is tha~ structural portion of an antibody m~leoule
com~ri~ed of a h~avy and light ohain vari~ble and
hypervariable regions that specifically binds
(immunoreaC~8 with) an anti~en. The term immunoreact in
its variou~ formg means ~pecific binding between an
antigenic de~erminant-con~alning molecule and a molecule
containin~ an an~ibody combining site such a~ a whole
antibody molecule or a portion thereof.
~ c~9slQ~ Db~ The phra~e monoclonal
antibody in its various grammatlcal forms re~ers to a
populatlon o~ antibody molecules that contains only one
~pecies of antibody combininq site capable of
immunoreac~ing with a particular antigen. A monoclonal
antibody thus typically display6 a single binding
affinity for any antigen with which it immunoreact~. A
monoclonal antibody may therefore contain an antibody
molecule having a plurality of antibody combining sites,
ea~ immunospecific for a dif~erent antigen, e.g., a
bispecific monoclonal antibody.
A monoclonal antibody i~ typically composed of
antibody molecule produced by clones o~ a single cell
suoh as a hybridoma that secretes ~produces) but one kind
of antibody molecule. The hybridoma cell is formed by
f~ing an antibody-producing cell and a myeloma or other
self-perpetuating cell line. Such antibodies were first
de~cribed by ~ohler ~nd Milstein, ~ L~ 256:495-4~7
(1975), which de~cription i~ incorporated by reference.
O c t . 1 ' ~ ~ 1 0 ~ D~3S S~r~1 ~; e r i e~ .S 1 S:~--5 q ~-- 1 3F~ 0 P . 1 1
i
~)00044
B. ~jgs~t~ Y~
As noted earlier, li~and-receptor complex
formation lie~ a~ the heart of many biological proces~e~.
Aside from the fact o~ the existence o~ such
intera~tion~, tho~e interactions lead to sub~equent
events that are manife6t in the biological processes to
whlch complex formation iB an initial step.
It has now been found that along with the
biologiaal function that accompanies ligand-receptor
formation, another, more sub~le change occur~. That
change i~ in the conformation of the llgand molecule that
results from the binding interaction with the receptor
and complex formation. That change in ligand
oonformation result~ in the ~ormation of a neo-antigen
that i~ ~xprqssed substantially on~y upon formation of
the co~plex. Ag no~ed earlier, that neo-antigen i8
referred to as a receptor-induced binding site, or RIBS.
The RIBS expre~ed by the complex formed by the
binding o~ fibrinoqen as ligand to GPIIb/IIIa as receptor
is used $11u~t~atively herein as exempl~ry of the RIBS-
forminq phenomenon. Monoclonal antibodies that
immunore~ot with the fibrinogen-GPIIb/IlIa complex but do
not ~ubstantially react with either the ligand or
receptor when not pr-s-nt in the complex are also
utilized herein as exemplary a~ anti-RIBS (or more
simply, RIBS) monoclonal anti~odle~.
In addition to the exemplary RIBS and RIBS
monocl~nal antibodies ~peci~ically discussed herein,
~everal additional ligand-rqc&ptor aomplexe~ are reported
in the literature. Tho6e complexes also form RIBS and
monoclonal antibodies can be raised to those RIBS, using
the techniques de~cribed herein. Those monoelonal
antibodie3 immunoreact with only the ligand portion of
the ligand-bound rec~ptor complex and not with the free
receptor or ~ree ligand. U6ing such RIBS monoclonals,
UC t . 1 ' ~ Y 1~ 1 U L~ ~ ~ ~r l ~ e r ~ e ~ c~ l Y~ ~ ~ W ~ . 1:~
Z~)00044
one can now a~say fer the presence and ~mount of a
ligand-receptor complex; i.e., an occupied receptor or
occupled li~and, in the presence of ei~her or both of the
free ligand and free receptor.
Exemplary, additional pair~ of RlBS-formin~
ligands and receptor~ ar~ ~umerated in ~a~le 1 that
follows. Theso pairs are a..o intended to be
illu~trative and non-limiting o~ the RIBS that can be
~ormed.
TABLE 1
~IGAND REC~P~O~ PAIR~
Ligand Receptor Citation
N~.
Von Wildebr~nd Factor GPIIb/IIIa
Vitronectin GPIIb/IIIa
Fibronectin Fibrone~tin
Recept,
ICAM-l LFA-l
Laminin CSAT 2
Collagen V~A-2 3
C3bi ~R3 Complement 4
Reaeptor
C3d CR2 Complement 5
Reoeptor
HIV-gp140 ~D4 T Cell 6
Receptor
FSH R~l~asing Proteln FRP Receptor 7
Apo B-100 Apolipoprotein 8
Re¢eptor
IL-2 Interl-ukin 9
Re¢eptor
Immunoglobulin~ Fc R~cep~or 10
Chorionic Gonadotrophin Somatost~tin 11
Receptor
PDGF PDGF Recep~or lZ
Oc t . 1 ~ ~2 13 e1~ GSSac1~1 se r i es ~i 19--5~-- 138 0 P . 13
1~0~()044
Tran~ferr$n Transferrin 13
Recep~or
1 Ruoslahti et al., Sc ~n~, 238:491-4~7 (1987).
5 2 Horwltz ~t al~, J, C~ Biol., 101:2134-2144
~1985).
3 Nieuwenhuis et al., ~ e, 31~:~70-472 (1~85).
4 Wrlght et al., Proc. Natl. Acad. Sci. US~,
84:1965-19Ç8 (1~87) ~
Nemerow et al.~ J. Yirol., 55:347-35~ ~1985) .
~ Guyader et al., Nature, 320:662-~69 (1987)~
7 Yu et al., Nature, 330:765-67 ~ 7).
8 Yamada et al., J. Clin. In~ç~t., 80:507 tl987).
~ Dower et al., Em~unol. Today, ~ 6 11~87).
Anderson et al., ~ YD9L~ 13~ ~ 2254 (1987).
11 Rim et al., J. Biol. Chem,, 262:470 (19~7).
12 Reating et al., ~ B~l. Chem, 252:7932 (lg87).
13 Kohqo et al., Blood, 70:1955 (1987).
~o ~. ~onoclonal A~ 9~Ç~
A monoalonal antibody tMAb) of the pre~ent
invention i~ characterized as comprising antibody
molecules that im~unoreact with a li~and's receptor-
induced blnding site.
A receptor-induced binding ~ite (RIBS) is a neo-
antigenic determinant that is expressed by a lig~nd when
the ligand 1~ bound by a receptor, bu~ i~ not expresQed
by the ~ree (non-bound) ligand. That is, a rcceptor-
ligand complex expre~ses a RIBS, but the non-occupied
30 receptor and the non-bound ligand do not. A MAb of the
present invention immunoreact6 with a RIBS, but does not
substantially immunoreact with either the unbound (free)
ligand or receptor, i.e., when either the ligand or
receptor 18 free in ~olution, and can therefore
distinguish between the reoeptor-bound and un-bo~nd forms
0~ t . 1 ' æ~ ~ ~GSS~ ser ie~ ~ 1~ - 54~ - l3æ~ P. 14
znow44
- 12 -
of a ligan~ becau~e it immunoreact~ with the receptor-
bound ligand but not with the fre~ ligand. The phra~e
"does not suhstantially immunoxeact wi~h either the
unbound ~free) ligand or receptor..." is utilized herein
~o ~ean that the monoclonal antibody-(llgand-re~eptor)
immunoreaction is inhibi~ed by no more than about 15
percent, and preferably less, by compe~itive binding with
free ligand or reaeptor~.
In one preferred embodlment, a subj~ct MA~
0 oompri~e antibody molecules ~hat immunoreac~ with a RIBS
expressed by a cytoadhesin-ligand complex. Cytoadhesin
is a name given to a super famlly of receptor molecules
all of Whi¢h ~ind to a ligand that contain~ the amino
acid re~idue sequence arginine-~lycine-aspartiC acid or
RGD. Plow et al., ~LQ~h_~at~. Acad. Sci. ~SA, 83:6002
(1~86). Thi~ ~uper family has also been given the name
Integrin. Rou~lah~i et 8l. ~ Sci~n~, 238;4~1-497 11987).
The particular cytoadhesin of exemplary inter~st herein
i~ GPI~b/IIIa~ al~o known a~ the platelet receptor.
A preferred monoolonal antibody of this invention
i~ ~ecreted (produced) by each of the following
hy~ridomas: hybridom~ 2G5 having ATCC deslgnation
HB9847, hybridoma 2F10 having ATCC de~i~nation HB9844,
hybridoma 3G11 having ATCC desiqnation HB9845, and
hybridoma 4G10 hav~ng ATCC de~ignation HB9846. The abovc
hybri~oma~ werQ deposited at th~ American Typ~ Culture
Collection, 12301 Parklawn Drive, Rockville, MD, 20852,
USA on Septe~ber 2~ 88 pur~uant to ~he Budape~t
Treaty .
D.
ComPoaitions
~he present lnven~ion contemplates a method
of forming a monoclonal antibody that immuno~eaot~ with a
reaep~r-inducod binding site. The method comprises the
~teps of:
Oc t . 1 ~ Q [;~C.S S:~I ~ e r i e ~ ci 19--5 ~ -- 13 f2 13 P . 15
2no0~)44
(a) Immunizing an animal with a receptor-
ligand complex. This is typically accomplished by
administering an immunologically effective amount, i.e.,
an amount sufficient to produae an immune response, of
immunogen to an i~munolo~ically competent mammal.
Pre~erably, ~he mammal is a odent ~uch as a rabbit, ra~
or mouse, although other man~al~ such as goat~, hor~es,
and simian~ can ~4 used. ~e mammal is then maintained
for a time period ~u~icient for the mammal to produce
0 c211s ~ecreting antibody mol~oules that immunoreact with
the receptox-ligand complex.
(b~ A suspenslon of antibody-secreting cells
removed from the immunized m mmal i~ then prepared. This
is ~ypically acoomplished ~y removing the splee~ of the
mammal and mechanically sep~ating the individual spleen
oells in a phy~iologically tolerable medium u6ing methods
well known in the art.
~ he ~uspended antibody produoing cells
are treated with a transforming agent capable of
producing a transformod ("immortalized"~ cell line.
Transformin~ agents and their use to produce immortalized
cell li~es are well known in the art and include DNA
viru6es such a~ Ep~tein Bar Virus (EBV), simian Virus 40
(SV40), Polyoma Virus and the like, ~NA viruses such as
Moloney Murine L~uk~mia viru~ (Mo-MuLV), ROUQ Sarcoma
Viru~ and the like, ~yeloma aell6 ~uch as P3X63-Ag8.~53,
Sp2/0-Agl4 and the like.
In preferred e~bodiment~, treatmen~ with the
tran~orming ag~nt rosults in the production of a
hybridoma by f~ing the suspend~d spleen oells with mouse
myeloma cells from a suitable ~ell line by the use of a,
suitable fu~ion promoter. The preferred ratio i~ about 5
spleen cells per myeloma ~ell. A total volu~le of about
108 ~pleno~yte6.
oc ~ . 1 æ~ ~ 44 ~e~13el DG~Sact~1 se~ ie~ ~ 54<~;-- 13~el P . 16
i2()0W~4
The cell line u~ed ~hould preferably be of the ~o-
call~d ~drug resistant" type, ~o that un~us~d myeloma
cell6 do not ~urvive in a sQlect$ve medium, while hy~rids
will ~urvive. The most common clA~ are 8-azaguanine-
refii~tant cell line~, which l~ck the enzyme hypoxanthineguanine pho~phori~o~yl tran~feraQe and hence are not
supported by HAT (hypoxanthine, aminopterin, and
thymidine) medium. It i6 al~o generally preferred that
the myeloma oell line us~d be of the ~o-call~d "non-
lo ~ecreting" type, in that it does not i~self produce anyantibody, although ecr~ting types may be used. In
certain cases, however, ecreting myeloma lines may be
pre~erred. Although the prefarred fusion promoter i3
polyethylene ~lyaol having an average molecule weight
~S from about 1000 to about 40~0 (commercially available as
PEG 1000, for example), other fu~ion promoters known ih
the art may be employ~d.
(d) The tran~ormed cell6 are then cloned,
preferably to monoclonallty. ~he cloning is preferably
performed in a ti~e culture medlum that will not
support non-~r~n~ormed c~ . When the transformed
cells are hybridomas, ~hls i~ typically performed by
diluting and culturing i~ separate aontainers, the
mixture of unfused spleen cells, unfused myeloma cells,
and fu~ed cells (hybxidomas) in a selective medium that
doe~ no~ support the unfused myeloma cells for a time
suffiaient to permit d~ath of the unfused oell~ (about
one week). The dilution and culturing is carried out in
~eparate oontainer6, and the dilution may be a limiting
dilution in whi~h the volume o~ diluent is ~tatistically
calaulated to i~olate a certain number of cells (e.g., 1-
4) in each separate container ~e.g., each well of a
miaroliter pla~e). The medium i~ one (e.g., HAT m2dium)
tha~ does not ~upport th~ drug-re~i~tant ~e.g., 8-
3~ azaguanine-regi~tant) unfu~ed myeloma cell line.
Oc t . 1 ~ a~ ~ ~5 0 ~ 3 [~CiSS~ ce r ie~ ~ 5~-- 1 3F:13 P . 17
20~ 4
~ 15 -
(e) The tlssue culture medium of the cloned
tran~formant~ i~ evaluated for the presence of ~ecreted
antibody molecule~ that do not immunoreact with a free
ligand but do immunoreact wi~ the ligand when it is
pre~ent a~ part o~ a receptor-ligand complex~ The
evaluation is performed u~ing well ~nown immunological
techniques, as i~ de~cribed hereinafter.
(f~ once a de~ired tran6~0rmant has been
identi~ied in ~tep (e), it is selected and grown in a
~uitable ti~ue ~ulture medium for a ~uita~le length of
time, followed by recovery o~ the desired ~ntibody ~rom
the aulture ~upernatant. The suitable medium and
~uitable length of cul~uring time are well known in the
art and are readily d~termined.
To produce a much greater concentration of
~lightly 1Q~ pure monoclonal an~ibody, the desired
hybridoma can be injected into mice or other mammal~ in
which the hybridoma can grow, pr~ferably 6yngeneic or
semisyngenic mic~. The hybrid~ma cau~e formation of
antibody-pr~du¢ing tumors a~ter a 3uita~1e inoubation
time, which re~ults in a high concentration of the
de~lred antibody (about 5-20 mg/ml) in the blood~tream
and peritoneal exudate (ascites) of he ho~it mou~e.
Media useful for the preparation of these
composi~ions are both well known in the art and
commeraially availa~le, and include ~ynthetic ~ulture
madia, inbred mi ce and the liXe. An exemplary synthetic
medium i~ ~ulbecao's minimal e~ential medium ~DMEM;
Dulbecco et al., Virol. 8:396 (1959)~ suppl~men~ed with
4.5 qm/l qluoo~e, 20 mm glutamine, and 20% fetal cal~
serum. An exemplary inbred mouae strain iB the Balbfc.
A ~onoalonal antibody produced by the above method
can be u~ed, ~or exam~le, in diagnostic and ~herapeutic
modalitie6, dis¢u~ed in grea~er detail hereinafter
whe~ein formation of a RIBS-containing immunoresction
Oc t ~ ; S~I = e r i e s ~ 5 4 ~-- 13~ 0 P . 1:3
2()~0044
- 16 -
product i~ de~ire~ Such u~e~ lnclude, for example, is
the diagnostic method~ and systems of the present
invention to detect fibrinoqen-bound platelets in a body
sample, ~or in vivo detection, of a throm~us, dispersing
a ~hrombu~, or thrombus imaging.
A RIBS monoclonal antibody is typically utilized
ln an aqueou~ compo~ltion. That compo~ition can be the
tissue oulture medium or asci~es fluid as obtained or in
dilute~ form. Such compo~itions are typically utilized
in v~o.
For in vivo uses, the RI~S monoclonal antibody is
typically puri~ied aQ l:y pre~:ipitation in ammonium
sulfate, af~inity purifica~ion procedure and the like and
then utilized in an agueous composition of a
pharmaceutically acceptable diluent. The concentration of
RIBS monoclonal antibodies in that aqueous compo~ition is
~d~sted to suite the desired use.
E. Therapeuti~ ~thods ~nd Composi~ions
Monoclon~l antlbodies secreted by any o~ the
before-descri~ed depo~ited hybridomas, and antibodies
having similar immunospecificity, are particularly useful
in areas relating to blood clo~ or thrombi. Thi6 is
because those types of anti-RI~S monoclonal antibodies
immunoreact with the fibrinogen-GpIIb/lI~a receptor
complex that i present on activatod pla~elets con~aining
bound fibrinogen, and platelet-bound fibrinogen is
involved in thrombus formation. Furthermore, ~nce by
definition, tho~e RIBS monoclonal antibodies do not
immunoreact with soluble fibrinogen as is present in ViYo
in circulating blood, ex~reme specificity of
immunorea~tion can be had by u~e of one or more of those
mono~lonal antibodie~.
1. Thrombus ~i~persment
Thus, one aspect of thi~ invention
contemplates ~ method for the dispersement of a thrombus.
:I c t . 1 ' ~ ~ ~3: 4 6 ~ G S ~:&~I s e r i e s ~ 19--5 4 '-- 13 ~ 0 P . 19
ZO~W44
- 17 -
Here, the an~ibody mo~ules of a monoclonal antibody
produced by at least o~e o~ t~e ATCC deposit~d hybridoma~
is chemically lin~ed to a pla~mlnogen-activating enzyme
~o form a conjugate in which the binding of the antibody
portion o~ the con~ugate to it6 RIBS i~ sub~tantially
unimpaired and the plasminogen activating activity of the
enzyme is substantially unimpaired. Method~ of preparing
antibody-protein conjugate in which the activities of
both portions of ~he conjugate are ~ub-ctantially
unimpaired are well known by ~illed wor~er~.
A plasminogen activating enzyme refers to the
group of fibrinolytic drug~ in the cla8s of
prot~rombolysic, ~uch as tissue pla3minogen activators
that induoe thrombolysis without systemi~ fibrinolysis or
fibrinogen break down. Other fibrinolytic drugs
contemplated are those which interact with the
proaotivator plafiminogen and include, bu~ are not limited
to 6treptokina~e, urokinas~ (u-PA), and tissue
plasminogen aatlvator (t-PA).
In ac~ordance with thi~ m~thod, a pharmac~utically
acceptable aqueou~ compo~ition containing a thrombu~-
di6persing amount of a before-de~cribed conjugate i5
admini~tered, such as ~y intravenous injection or
infusion, to a mammal ~uch as a human having a thrombus
to be dispersed. ~he mammal oo treated (administered
mammal) is maintained for a time period ~ufficient for
the antibody por~ion of the conjugate to immunoreact with
the platelet-bound ~ibrinogen complex pre~ent in the
thro~bue and for the pla~minogen-activating enzyme
portion of the con~ugate to activate pla~minogen. Since
removal of th~ conjugate would ~e a dif~icult and time-
con~uming ~a8X, the treated mammal is maintained for a
time sufficient for its own body to clear the conjugate
by u~ual means.
Z. Inhibi~ion of Thrombus Formation
O c t . 1 ~ G S S~c~l . , ~ ~P r i e s cS 1 9--5 ~ 6-- 1 3 8 ~3 P . ~ el
ZOaO~44
- 18 -
Another treatment method is contemplated that
i~ u6eful for animal ~ubjects that are at ri~k of
thrombu3 formation ~uch a~ per60ns in the ~ir~t several
day~ after a ~a~or operation like a coronary by-pa~s
operation.
Here, a ~hrombu~-inhibiting amount of a monoclonal
~ntibody cont~ining antibody mocules produc~d by at lea~t
on~ of the ATCC deposited hybridomas 2G5, 2F10, 3Gll or
4C10 present in a pharmaceutically ~oeptable aqueous
aompo61tion iB administered to a mammal such as a human
in whom thrombua formation is to be inhibited. The
treated mammal (administered mammal) i~ maintained for a
time period ~uffiaient for the administered RIBS
monoclo"al an~ibodies to be cl~ar~d from its body by
usual mean~.
In this method, the immunoreaC~ion of the RIBS
monoclonal an~ibodies with activated platelet~ containing
bound ~ibrinogen inhibit& thromb~s for~ation. Of course,
since a RIBS mono~lonal antibody immunoreact~ with the
fibri~ogen-GpIIbJIIIa complex on the platelet and not
wi~h fibrino~n in the blood, normal func~ion of th~
treated anim~l is not impaired.
In a related embodiment a method of inhibiting
platelet aggrega~ion is con~emplated that compri~es
administering to a platelet containing solution a
pnarmaceutically acceptable aqueous composition
containing monoclonal antibody molecules produced by at
leas~ one of ~he ATCC deposited hybridomas 2GS, 2F10,
3G11 or 4C1~ A platelet aggregation-inhibiting amount
of the antibody i8 admini~tered in vivo to a animal
subject in which inhibition of platel~t aggregation is
de~ired, or is admixed in vitro with a platelet-
containing ~olutlon, 6uch as plasma or blood. The
immunoreaction of the RIBS monoclonal antibodies with
f~brinogon-GPIIbJIIIa complex on the platelets forms an
DC t . ~ ~ el 5 ~F~ ell~telel ~13~:5aC~1 ~er ies ~ 5~$-- 138la F~ . ~ 1
Z~ 44
-- lg --
immunoreaction prodUat that inhibit3 platelet
aggregation.
A 6ingle, before-de cribed, RIBS monoclonal
a~ti~ody can be u~ed in each of the aboVe methods, or a
mixture con~aining more ~han one can be utilized. Thus,
although each o~E the monoclonal antibodies produced by
the four ATC~ deposit2d hybxtdoma~ sh~res the property of
being a RIBS monoclonal, each an~ibody combining site
does not immunoreact with ~he ~ame epitope. Thus,
o advant~ge can be had by using a mixtur~ of those
mono~lonal antibodiefi ~alonq or 1n the conjugate) so that
multiple bindlng of the aombining sltes to a single bound
fib~inogen molec~le can be had.
I~ is to be under~tood that although tne two above
methods have b~en described in terms of the RIBS
monoclonal antibodie that immunoreact specifically with
the fibrinogen-GPIlb/IIIa complex, similar methods are
applicabl~ for other ligand-receptor aomplexes that
con~ln RIBS.
The preparation of a pharmaceutically accep~able
aqueous composition that contain~ antibody molecules as
active in~redients is well understood in the art.
Typiaally, such compositions are prepared as injectables,
either as liq~id solution~ or su~pen~ions, however, solid
form6 suitable for solution in, or suspen~ion in, an
aqueous liquid prior to in;ection can al~o be prepared.
~he preparation can al~o be emulsified.
~he conj~gate or monoclonal antibody alone is
often mixed with sxcipient~ that are pharmaceutically
aaceptable and ~ompatible wltn the aon~ugat~ or
monoalonal antibody a~ are well known. S~table
excipien~ are, for example, water, saline. dextro~e,
glycerol, ethanol, or the like and combi~ations thereof.
In addition, lf de~irsd, the composltion can contain
~5 minor amoun~s of auxiliary substances such a~ wetting or
Oc t . 1 ~ 85' ~: 45:~ lal3~ DGS~c~l se r ies ~ 15~--54~-- 13~0 P ~ 22
2000044
- 20 -
emul~ifying agents, pH bu~ering agents whic~ enhance the
effeativenes~ of the aç~ive ingredient.
A conjugate or monoalonal antibody can ~e
formulated into the a~ove aqueo~ff composition as
neutralized pharmaceutically accepta~le salt forms.
Pharmaceutically acceptable ~alts inc~ude the acid
addition 5~1t5 ( formed with the free amino group~ of the
enzyme or antibody ~olecule~ ~hat are formed with
inorganic acids ~u¢h as, ~or example, hydrochloric or
lo phosphorla acid~, or ~uch organic acids a~ acetic,
tartaric, mandelic, and the like. Salts for~ed wi~h the
free carboxyl gro~ps can also ~e derived from inorganic
bases such a , ~or example, sodium, potas~ium, ammonium,
calaium, or ferric hydroxide~, and ~uch organic ~as~s as
isopropylamin~ trimethylamine, 2-ethylaminoethanol,
hi~tidine, procaine, and the like.
The therapeutlc antibody molecule-aontaining
compo~itionS a~e conventionally admini~tered
intravenou~ly, as by injec~ion o~ a uni~ dose, for
example A composition i~ administere~ in a manner
compatible wi~h the dosage ~ormulation, and in a
therapeutically offective, i.e., in a thrombus-dispersing
or thrombus-~nhi~iting, ~mount.
The quantity to be administered dependæ, in~er
alia, on the an~mal specie~ to be treated, the subj~ct
animal's 6ize, the size of the ~hrombus (~f known), the
amount of fibrinogen-bound platelets present, and
capacity of the ~ubject to utiliz~ tne con~ugate or
monoclonal antibody. Precise amountR of conjugate or
monoclonal antibody required to be adminitar~d depend on
t~e judgment o~ the practitione~ and are peculiar to each
individual, particularly wher~ human6 are the trea~ed
animal~. Dosag~ r~ngeæ, however, can ~e characterized ~y
a therapeutically effective blood concentration and can
range from a concentration of antibody-containing
~ c t . 1 ~ :3 5 la: 4 5~ 13 e~ t3 e~ G 5 S~ 'l ~ e r i e s ~ 1~--5 cl ~-- 1 3 F: 13 p, z 3
2~)00044
- 21 -
conjugate or antibody alone of the present invention from
about O.01 uM to about 100 uM, preferably about o.1 uM to
lOuM.
Suitable re~ime~ for initial ad~ini6tration and
boo6ter injections are also varlable, but are typi ied by
an initial administration followed by repeated doses at
one or more hour interval~ by a ~ubsequent injeotion or
other admini~ration. Alternatively, continuou~
intravenou~ infuslon sufficient to maintain
therapeutically e~fective concentrations in the blood are
cont~mplated.
F. Di~ ~
A dlagno~tic system in kit form of the
pre6ent invention includes, in an amoun~ sufficient for
at least on2 assay, a RI~S monoGl~nal an~ibodies of the
present invention, such ~s one produced ~y one of the
~our A~C dRposi~ed hybridomas, a~ a separately packaged
reagent. A label for ind~ating the presence of an
immunoreaction ~etween the RIBS and RIBS monoclonal
antibody ig also pr~erably included in the same or a
separate package. Instructions for u~e of the pacXaged
~eagent are al60 typically included.
Instructions f~r u~e typically in~lude a tangible
expres~ion de~criping the reagent concentration or at
z5 lea~t one a~say m~thod parameter such a8 the relative
amounts of rea~en~ and ~ample to be admixed, maintenance
time periods for reagent/sample admixtures, temperature,
buffer condition~ and the like.
one embodiment of this invention i~ a diagnostic
system in kit form for assaying for fibrinogen-bound
platelets in a platelet-oontaining va~cular fluid ~ample,
suoh a~ ~lood or pla~ma. The sy~tem compri~es a package
containing a monocl~nal antibody that immunoreacts with a
RIBS ~xpres~ed by ~he reeeptor-ligand compl~x.
Preferably, th~ anti-RIBS antibody molecules of the
~1~ t . 1 ~ 85~ ~: 5 ~ ~ ~GS5~1 ce r i ec .S 15~--54~i-- 1~8 0 P . 24
ZO`OW~4
- 22 ~
monoclonal an~ibody are tho~e produced by one o~ the
following hybrldoma~: hybridoma 2G5, hybridoma 2~10,
hybridoma 3Gll, and hy~ridoma 4Glo. Preferably, the
antibody molecules are present a~ a monoclonal antibody
composition which contains more than one particular
monoclo~al antl~ody. Further preferred are kits wherein
~he antibody moleoules are lin~ed ~o a radionuclide
label, prefera~ly a 125I-labeled or other labeled antibody
mole~ule~. U~eful labels are di~cussed hereinafter.
lo In another embodlment, a diagno~tic ~y6tem of the
pre~ent inven~ion is suitable ~or assaying ~or the
presenoe of a thrombus ~ vivo. The sy~tem comprises a
package conta~ning monoclonal antibody molecules that
immunoreac~ with a ~IBS expre6~ed by a receptor-ligand
complex. Preferably, the antibody molecule~ present are
those ~ecreted by a hybridoma selectRd from the group
consistlng of 2G5, 2F10, 3Gll, and 4G10. The antibody
molecules prefera~ly lin~ed to an in vivo label or
indicating means.
Although a kit for i~ vivo imaging can often be
utili~ed for in Yi~LQ assays, it is understood that the
converse need not ~e true. For example, the monoclonal
antibodies u~ilized for ~a_YiYQ worX 6hould be free of
pyro~en~ as should any buffer salts of ~queous
compositiOns and reagents. Freedom from pyrogen content
i~ not a necessity for in vitro assays. Additionally,
the indic~ting means u~eful for in vivo imaging is
typically different from that used i~ vitro, as i~
discussed hereinafter. Thu~, the a~ay system is
"suitable" for in_Ya`Y2 imaging, and "suitable" buffer
salts, aqueous solution~ and indicating mean~ can be
supplied as part of the kit in the ~ame or separate
packages.
In preferred embodiment~, a diagno~tic system of
t~e presen~ i~vention further includes a label or
::lc t . 1 ' ~:9 la: 5 1 1313 0 el GGS~;acl`'l ~e r i es ~ 54cS-- 13~ ~ P . ~5
Z000044
- ~3 -
indicating mean~ capable of signaling the formatlon o~ a
complex containing an antibody ~nolecule of the present
invention.
A~ ueed herein, the terms label and indioating
means in their v2rious gram~atical form~ refer to single
atom~ and moleculea that are either dirsctly or
indirectly i~volvcd in the produc~ion of a deteotable
signal to indicate the pre~ence of a complex- In.
labels or indicating ~ean~ are tho~e u~eful ~ithin the
body of a human ~ubjec~ and include ~1~In~ ~c, ~Ga,
, 1~2I, and ~ ndium-
Any label or indicating means ~an be linked to or
incorporated in an antibody molecule that is part of a
~on~ugate or monoclonal antibody compo6ition of the
present lnvention, and those atoms or molecules can be
u~ed alone or in conjunc~ion with additional reagents.
Such labels are themselve~ well Xnown in clinical
diagnostic ohemistry and congtitute a part of this
invention only in o~ar as ~ney arg utilized with
otherwi~e novel proteln method6 and~or sy~tems.
The llnking o~ labels, i.e., labeling
of, to antibodiee i~ well known in the art. For
in~tance, antibody molecule~ produced by a hybridoma can
~e labeled by mctabolic lncorporation of radioisotope-
containing amino acids provided a~ a component in theaulture medium. See, for example, Galfre et al., ~t~.
~Y-YLL~ 73:3-46 ~1~81). The ~echniques of protein
conjugation or aoup~ing ~hrough activ~ted functional
group~ are parti~ularly appli~able. See, for example,
Aurameas, et al., ~ , Vol. 8 Suppl. 7:7-
23 (1978), Rodwell et al., ~1~ ech., 3:889-894 (1~84),
and U.S. Pat. No. 4,4~3,795.
~ he in vitro diagno~tic ~y~tems ~an al80 inelude,
pref~rably as a ~epara~e pacXage, a speairic binding
3~ agent. A ~pecific blnding agen~" i a molecular ~ntity
~c t . 1 ' 85~ ~: 5 1 01~ ~GSSa~1 ~e r i e~ _ ~ 9--54 :S-- 1 3f:: 13 ~ . 2c;
04~
- ~4 -
capable of selectively binding A monoclonal antibody of
the present inven~on ~ut is not itself an antibody
molecule of the presen~ invention. Exemplary ~pecific
bindin~ agent~ are 6econd antibody molecule~, complement
proteins or fragments thereof, ~ _3gLys protein A and
the li~e. The specific ~inding aqent binds the RIBS
monoclonal an~ibody mol~cule of this invention when that
monoclonal antibody i8 pre~ent a~ part of an
immunocomplex with ~he llgand-receptor complex. In
preferr~d emb~diments, the ~pecific binding agent is
labeled. However, w~en the diagno~tac 6y~tem include~ a
~pecific blnding agent that i8 not la~eled, the æpecific
binding agent is ~ypically used as an a~plifying mean~ or
reagent and a second reagent tha~ is labeled bind~ to the
specifi~ binding agent (amplifying means). In these
embodiment~, the labeled second reagent i~ capable of
specifically ~inding the amplifying means when the
amplifying m~an~ i6 bound to a RIBS monoclonal antibody-
contalning immuno~omplex.
~he diagnostlc kits of the present invention can
be used in ~n "ELISA" format to deteat the presence or
quantity o~ ligand-reaeptor complex uch aæ fibrinogen-
bound platelet~ in a body fluid sample ~uch as serum,
plasma or urine. "ELI~A" refers to an enzyme~linked
imm~n~orbent assay that employs an an~ibody or antigen
bound (here, the RIBS-containing ligand-receptor comple~)
to a 601id phas~ m~trix that forms a aolid support and an
enZyme-antigen or enzyme-antibody cunjugate to detect and
guantify the amount of an antigen or ant~body present in
a ~ample. A desc~iption of the ELISA technique ~ found
in Cha~ter 22 of the 4th Edition of
~Lunoloqy by P.P. Sit~s et al., publi~hed by Lange
M~dical Pu~lication~ of Lo3 ~lto~, CA in 198~ and in U S.
Patent~ No. 3,654,090; No. 3,850,752; and No. 4,016,043,
which are all incorporated h~rein by reference.
O c t . t ~ 5 2 e~ D G S S~ 1 ~ e r i e s <~ 5 4 ~ ~ 1 3 8 0 P . ~ 7
Z~)0004~
-- 25 --
In pre~erre~ ELISA kit embodiments, the antibody
mole~ul~s of ths pre~ent invQntion are affixed to a 6011d
matrix to form a ~olid ~upport that is ~par~tely
pac~aged in the ~ubjeat diagnos~ic 6y~tems. The
antibodie~ are ~ypioally affixed to the 601id matrix by
adsorption ~rom an aqueous medium alt~ough other modes of
affixation, well known to those skilled in the art can be
usQd.
A labeled spRoific binding agent that binds to the
o aomplex or to it~ constituents or an unlabeled speciflo
binding agent plu~ ~ la~eled second reagent that binds to
the ~peai~ic binding agent is al~o included in one or two
separate pac~ages, re6pe~tively, in the ~it. using one
o~ the mono~lonal antibodies produced by one o~ the A~CC
depoæited hybridoma~ as exemplary of the matrix-bound
antibody, labeled anti-fibrinogen antibodies as are
commeroially available are exemplary of the spe~ific
binding agent.
Useful solid matrices are well known in the art.
~o Su~h materials include the cro6s-linXed dextran available
under the trademark 5~PHADEX from Pharmacia Fine
Chemicals (PiecataWay, NJ~; agarose; bead~ of polystyrene
bead~ about 1 mi~ron to about 5 mlllimeters in diameter
available from Abbott Laboratorie3 of North Chicago, IL;
polyvinyl chloride, polystyrene, ~ros6-linked
polyacrylamide, nitrocellulose- or nylon-~ased webs such
as sheets, strips or paddles; or tube5, plates or the
well6 of a microliter plate ~u~h as those made from
polystyrene or polyvinylchloride.
The monoclonal antibodie6 (labelsd or unlabeled),
labeled ~peci~ic binding agont or ampli~ying reagent of
any diagnosti~ ~ystem described herein can be provided in
~olution, a~ a liquid dispersion or a6 a sub6tantially
dry power, e.g., in lyophiliæe~ ~orm. Where the
indi~at$ng mean~ is an enzyme, the enzyme's substrate ~an
~c t . 1 F~ 5~ 3 ~ 0 c~Gss~r1 ~e r i e~ ~ 54~-- 13813 P . ~
ZS~00044
- 26 -
also be provided in a ~ep~rate package of a kit system.
A solid ~upport matrix such as the be~ore-de~ari~ed
microl~ter plate and one or mor~ bu~fers can also ~e
included a~ separately packaged element~ in this
dlagnostic as6ay system.
The packages dl~aussed herein in rela~ion to
diagnostic systems are ~hose customarily utllized in
diagno~tic systems. Such package~ include glass and
plastic (e.g., polye~hylene, polypropylene and
poly~arbonate) bottle~, vialQ, plas~ic and plas~ic-foil
laminated envelopes and the like.
G. ~
The present inventlon contemplates a method
for detecting a receptor-ligand complex, as, for example,
is fo~nd in a thrombus or in fibrinogen-bound pla~elets,
preferably, GPIIb/IIIa:fibrinogen. The method utili~es
~he expresgion of a receptor-induced binding site (RIBS~
and a monoclonal an~i~ody molecule ~hat immunoreacts with
the RI~S in the ligand portion of the receptor-ligand
aomplex, but doe~ not reac~ with a non-bound recep~or or
a non-bound ligand. ~hose ~killed in the art wlll
under~tand ~hat thera are numerous well known clinical
diagno~tic cheml~try procedu~efi tha~ can be utilized to
form tho~e immunocomplexes. Thus, while exemplary assay
methods are de~cribed herein, the inven~ion i8 not so
~imitod.
1. Lll~
More specifically, a method for detecting ~e
presence o$ a thrombus i~ a mammal such as a human is
~ontemplated. An aqueous co~position containing an
imaging-effective amount of a monoclonal anti~ody of the
pre~en~ invention con~aining antibody molecules lin~ed to
an in vivo indicating m~ans is intravenously administered
to a ma~mal ~uch a~ a human in need of ~uch treatment.
0~= t . 1 ~ 5~ ~ D~SS2~1~1 ce r i es ~ 1~--54~-- 13~: 0 P . ~:~
Z000~4~
-- 27 --
The admini~ered ~ammal i8 maintained for a
predetermined time period suffi~ient for the labeled
antibody molecul~ to immunoreac~ with the platelet-bound
fibrinogen oomplex present as part o~ a throm~us. The
~ubject mammal iB then ae~ayed for the pre~ence and
prefe~ably loaation of any labeled immunocomplex formed,
and there~y detect~ the thrombus and i~ location. Since
the labeled RIBS monoclonal antibodies normally contain
radionucl~de~ a~ the label or indicating means, ~he
0 imaging asBay is oarried out by u~ual, well known
radioi~aging technigues. Such techniques can distinguish
the relatively high concentration of radiolabel at the
thrombus from the relatively lower systemic amount of
radio label. Where relatively long-lived radioisotope
are utilized, the administered mammal ~an be ~aintained
for a tim~ period 6ufficient for sub6tantial systemic
clearanoe of the labeled mon~alonal antibody to thereby
provido a rela~ively ~till lowor amount of background
~i~nal from the radiolabel.
z,
~ ariou~ heterogeneou~ and homogeneous assay
protocols Gan be employed, either competitive or non-
competitive, for detecting the pre~ence and preSerably
amount o~ fibrinogen-bound platglets in a platelet-
containing and/or free fibrinogen-containing ~ody sample,
preferably a body fluid ~ample such a~ blood or a
platelet-containing portion of blood. For example, a
heparin-preserved (non-clotted) blood sample and an 125I-
labeled form of one o~ the before-di~cus~ed, deposited
RIBS an~ibody molecule~ are admixed. Amounts and
concentrations of sample and labeled monoclon~l antibody
are, of courge, utilized so ~hat a meaningful refiult ~an
~e obtained. The immunoreaction admixture thus ~ormed is
maintained under biological assay condition~ for a time
~c ~ . 1 æ~ ~ 5~ ~ G55~1~1 ser i ec ~ 1~--54~-- 13~: 0 P . 30
ZC~ )4~
- 28 -
period ~ufficient for fibrinogen-bound platelets pr~sent
in the ~ample to immunoreact with the labeled antibodie~
and form a labeled immunoreaction product. ~he labeled
immunoreactio~ produat, when present, is then separated
from any non-reacted labeled-antlbodies that may be
present. In homogenou~ a~ays, ~eparatiOn i~ typically
by centrifugation suffi~lent to pellet all platelets
pre~ent in the gampla. In heterogeneous assay~ such as
an ELISA, the immunoreaction prod~ct i~ ~ound to ~he
~olid ~upport and the ~epara~ion is typically effected by
a washing 6tep in which any unbound RIBS antibody 1~
discarded and th~ solld support-bound immunoreaction
produc~ i~ r~tained.
It should be understood that where an unla~eled
RIBS monocl~nal antibody is used to immunoreact with a
RIBS-containing ligand-receptor complex, a ~econd
admixtu~e i~ formed b~tween the before-described,
separated immunocomplex and a labeled binding reagent or
unlabeled bin~ing reagent u~ed as an ampllfYing means.
That reaction mixture is maintained under biological
conditions for a time period ~uf~icien~ for a ~econd
binding complex ~o form between the first-formed
immunocomplex and the speci~ic binding reagent. (Where
the specific biding reagent compriges antlbody molecules,
that second binding complex is a ~econd immunocomplex.
Where ~. aur~u~ prot~in A i8 used, for example r the
second binding complex does not rely for binding upon the
antibody binding site, and that complex is best referred
to as a ~inding complex. since an immunocomplex i8 a
sp~cific binding aomplex, the complex formed between the
specific binding r~agen~ and first-named immunocomplex is
a second binding complex.) The second binding comple~ is
th6reafter ~eparated from any unreacted sp~ciflc ~inding
~eagent that may be pre6ent, a~ by a previou~ly mentioned
technique, and the presence of the la~el and th~reby
O c t . 1 ~ æ ~ ~: 5 5 ~ ~ S 5~ e r i e s ~ 5 ~ 6-- 1 3 8 0 F~ . 3 1
Z~)()0044
- 2~ -
immunocomplex i~ determined, and prefera~ly quanti~ied.
Wh~re ~he epecific binding reagent is not labeled
and i6 used as an amplifying means, a third admixture is
formed from ~he above, separated second biding ~omplex,
5 when present, and a label-containing second binding
reagent. The presenae o~ the label in the second binding
complex is, of cour~e, not determined in the above-
re¢ited method where no labeled reagent Wa~ admixed. The
before-described maintenance and separation steps are
~o repeated for this aspect of the mothod with a label-
contalning third b~nding complex ~eing f~rmed and
retained, The pre3ence and preferably the amount of the
label ie ther~after determined.
ThU~, in each of ~he above-de6cri~ed aspeots o~
thi~ assay, the presence and preferably the amount of the
label provldes the ~asis for det~rmining the presence and
preferably th~ amPunt of fibrinogen-bound platelets.
It is noted that the worker aarrying out an above-
de~cribed assay usually will not know whether one or more
of the immunocomplex or bindinq complexes hae indeed
formod until the amount of la~el present i~ determined.
Ne~ertheless, all o~ the steps of a given as~ay procedure
are carried out as i~ the R~S-containing aomplex were
indeed present.
It i~ to be emphasized that b~cause of th~ unique
~pecificity of ~he RIBS monoalonal an~ibodies, the abovQ-
de&cri~ed a~say for fibrinogen-bOund platelets and the
~efore-de6crl~ed imagi~g ae~ay ~or a thrombus can be and
preferably are ~arried out in the presence of both free
platelets and free fibrinogen, i.e., non-complexed
platelets and fibrinogen. As a result, special handing
pro~edure such as.separations and washin~ steps need not
be carried o~t on the body #ample prior ~o U6~ of that
sample in an assay. ~hls ~eature is common to all assays
using RIBS monoclonal antibodies.
O c t . 1 ' 8 S~ 5 ' e1 1~ ~ S S~ 1 s e r i e s ~ 1 S~--5 ~ ~-- 1 3 a 1~ P . 3 :~
Z~)~0044
- 30 -
Biological assay aonditions ~re tho~e tha~
maint~in the ~iologica~ activity of the antibody
molecules of ~hi~ invention and the fibrinogen-bound
pla~elets or other R~BS-containing complex 60ught to be
assayed, Tho~e condi~ions include a temperature range of
about 4 degrees ~ to about 45 degrees C, preferably about
37 degrees C, a pH value range of about 5 to about 9,
prefera~ly about 7 and an ionic strength varying from
that of distilled water to t~at of about one molar sodium
ahlox~de, preferably a~out that of physiological ~aline.
M~thods for optimizing euch conditions are well known in
t~e ~rt.
~LES
The following example~ are intended to illustrate,
~ut not limit, ~he preaent invention.
1. ~y~L~doma and Mono~lonal AntibodV Production
~onoclonal antibodies were produced using
6tandard hybridoma technology. sriefly, Balb/c mice were
immunized and 6ubsequently boosted three times with about
50 micrograms ~ug) per mouse of ~ibrin D-dimer immunogen
prepared sub~tantially a~ de6arlbed in CiernieW3ki et
al., hromb. H~emQ~tas., 48:33-37 ~1982).
Subseguantly, 1.23 X 108 6plenocyte cell6 from one
immunized mou~e whose antibodie~ immunoreacted with the
immunogen were admlxed with 2.46 X 107 P3Ag8653.1 mouse
myeloma cells in the pre~once of the cell fusion promo~er
polyethylene glycol 4000. The antibody producing aell~
thu~ transformed were tran~ferred to 96-well microtiter
plates at a deneity of about 3 X 104 cells per well and
culturad.
Ti~sue culture ~upernatants from 235 wells
appearing to oontain viable hybridoma~ after about 14
days of culturing were s~reened by radioimmune a~say for
the presence o~ anti-RI~S antibody ~olecules. Briefly,
100 microliter6 (ul) of phosphate-buffered ~aline (PBS)
Oc t . 1 ~ 5~ ~ 355a~1 ~ e r i e~ e. 1 $--5 4 ~s-- 1 3s~ 0 p 3 ~
20boo44
- 31 -
containing ei~her 1 ug/ml of fibrinogen or low density
lipoprotein ~LDL) (control) w~r~ admixed into t~e wells
of flat-botto~ Y6-well polyvinyl microtiter plates as
solid phase matrix. The plate~ were then maintain~d for
about 16-20 hours at 4C ~o permit ~he fi~rinogen or LDL
to adsorb onto th~ surfaae of tbe wells to form a solid
~upport. The coating solution was removed by shaking,
~he wells were rin~ed, and 100 ul o~ blocklng solution
(PBS containing 5% normal qoat C~rum) were admixed into
Rach well to block exce~s protein ~inding 6~te~.
The wells were maintained for about 30 ~inutes at
37~C and then the'blocking solution was removed. Into
oach well were ~hen admixed 100 ul of either (a)
hybridoma tis~ue culture superna~an~ diluted 1:10 in PBS
or (b) hy~ridoma supernatant diluted 1:10 in PBS
containing 100 ug/ml fibrinogen as a competitive
inhibitor. The immunoreac~ions admixtures thus for~ed
were maintalned at room ~emperature for about 1~-~0 hours
at 4'C to permit the formation of a solid pha~e-bound
immunoreaction product and a llquid pha~e, including any
non-bound monoclonAl antibody molecules.
To each well were then admixed 100 ul of 12sI-
labeled goat anti-mou~e IgG. The labeling immunoreaction
a~mixture thu~ ~ormed was maintained about 6-20 hours at
40C to pærmi~ ~ormation of a 12sI-labeled ~econd solid-
phase im~unoreaction product. The solid and llquid
phases were separated to remove any non-bound lZ5I-goat
a~ti-mou~a IgG. Th0 amoun~ o~ ~Z5I-bound to eaoh well was
determ~ned by gamma solntillation.
The pr~senCe o~ at least about 3 times the amount
of non-specifically bound 1Z5I, as determined from the
LDL-coatod well~, in a fibrinogen-coated well indicated
~he presence of anti-~ibrinogen antibodie~ in a tissue
culture super~atant. A reduction of solid-phase bound
125I by no more ~han aPout 15~ by the presence of liquid-
Oc t . 1 ~ 57 ~ 0 ~3 DGSSa~1 se r i esi ~ 15~--5~5-- 138 0 P . 3~
200~044
- 3a -
phase fibrinogen competitor in the immunoreaction
admixt~re [part ~b) above] indicated the presence of an
anti-RIHs an~ibodies in the tissu~ culture supernatant.
The above ~creening procedure resulted in the
~dentificatlon of 4 hy~ridoma~, designated 2G5, 2F10,
3Gll and 4G10, that produce anti-RI~S antibodies that
~nd a fibrinogen:GPIIb/IIIa ~omplex.
Each of th~ four a~ove d~scri~ed hybridoma~ was
cloned twice by limitin~ dilution and sub~equently used
to produce a~aites fluid. The antibodie~ wer~ then
isol~te~ from the asaites fluidR by using protein A
S~pharo~e~
Compo~it$ons containing Fab ~ragm~nts of the
monoclonal antibody (Mab) 2F10 were prepar~d by digestion
of protein A Sepharo~o - 1~olat~d Mab aF10 with papain
(200:1 w~igh~ per weight of Ma~ to papain) for ~ hours at
37C following the methods o~ Mage ~t al., ~~h~ in
~nzy~Llcly~ 70: 142-50 (1980). Undi~e~ted antibody and
Fc fragments were removed from the ~ab frag~ent~ by
ahromatoraphy on protein A Sepharose. The re~ultin~ Fab
fragments were collected from the Separose to form a 2Flo
Fab preparation.
2. Detection of a Fibrinooçn:GPIIb~IIIa RI~S on
~onoclonal antlbodies produced by hybrido~as
2G5, 2F10 and 3Gll, i.Q. MAb 2G5, MAb 2F10 and MAb 3G~,
rsspect$vely, were examined ~or ~heir ability to
immunorea~t w~th a cell ~ur~ace bound RIBS. Each of the
~our monoalonals was 125I lab~led ~ing tandard
Chloramine-T methodology. Greenwood et al., Bio. Chem.
J., 89:114-123 (1963).
Sixty milliliter~ (ml) of whole human blood was
colle~ted in 5 ml of A~D (0~065 M citric aeid, 0.085 M
~odium citrate, a~ dextro6e) containing hlrudin ~Sigma
Chemical Co., St. Loui~, M0) at a formula conoentration
Oc t . I ' ~ 8: 5~ ~3 la 0 ~ G:~;S~r~l ~e r i e~ ~ 19--54 li-- 138 0 P . 35
~000044
- 33 -
of 0.06 units per ~1 (U/ml) and centrifug~d for 15
minuteC at 120 x g. The resul~ing supernant~ de~ignated
plat~let rich plasma, wa~ xecovared, isolated and further
centrifuged ~or 15 minutes at 1200 x g to form a pellet
of isola~ed platelets.
The i~olated platelet~ were resuspended in 2 ml of
calcuim-fr~e Tyro~es buffer ~0.13 M NaCl, 0.00~6 M KCI,
0.002 M MgC12-6H20, 5mM Hepec~ 0.012 M NaHCO3, pH7.2)
containing 1 mg/ml bovine se~um albumin (BSA) and 1 mg/ml
o dextrose. The platelet SUBpension was then applied to a
Sepharose CL2B column ~40 ml total bed ~olume; Pharmacia
In~., P~sc~taway, ~J) equilibrated wi~h the same Tyrodes'
buf $er . Was~ed platelets were recovered from the void
volume of the CL2B column in a ~inal volum~ of about 4 to
5 ml.
Sample~ of the wa~hsd platelet~ were then
stlmulated (aGtlva~ed) by admixture with eith~r adenosine
dipho~phate (ADP) to concentration of lo miaromolar (uM),
or thrombin ~o a concentration o~ 0.1 units/ml. some
sample~ o~ ADP-stimulated platelets were also admixed
with fibrinogen tD a ~ibrinogen conçentration of l mM.
To each sample of platelets, including some non~
slmulated control ~amplea, was admixed a ~25~-labeled MAb
to a concentration of lO nanomolar (nM~. The
immunoreaction ~dmixture thus formed was maintained for
30 minutes at 22-C to p~rmit immuno~eaction product
~ormation. The immunoreaction products were separated
from non-bound 1Z5I-MAb by centri~ugation throug~ 0.3 ml
of 20~ sucrose. The ~mount Or 125I-MAb assoc~ated with
the platelet pellet wa determined by s~in~illation
spectrometry.
The re~ult~ of this study, shown in Table 1,
indicate that the anti-RIBS monoclonal antibodies do not
6ubstantially immunoreact with non-stimulated platelets.
~ow~ver, when the platelet~ are ~timulated with an
2000(~4~
- 34 -
agoni~t such as ADP or thrombin, a siqniflcant
immunoreaction of the MAbs with th~ fibrinogen:GPIIb~IIIa
complex on the aell~ i8 obtained. Stimulation of the
platelet with ~P or thrombin rexults in secret~on and
~urfacs binding by GPIIb/IIIa of the platelet-e~dogenou~
fi~rinogen. Ad~ition of exogenous fibrinogen did not
neutralizR (inhibit~ the binding of 125I-M~b to the
stimulated platelets, thus indicating ~he MAbs are RIBS-
specific~ i.e., they do not immunoreact with free
fibrinogen ln ~olut~on. Si~ilar re~ult~ were obtained
with MAb 4G10.
TABLE l
~ MAb ~ound
(cpm~plat~let3
Platelets 2G5 ~FlO 3Gll
Non6timulated 1,200 2,800 1,300
ADP-stimula~ed 33,750 43,~00 31,550
ADP-stimulated +
fibrinogen 35,600 51,300 30,150
~hrombin-
stimulated 28,620 ~2,000 28,400
To emphasize the point that exogenou~ly
added fibrinogen i~ not cell-associated (i~e., is not
par~ of cell receptor-li~and oomplex) but yet does not
neutralize ~he immunoreactivity of MAbs ~G5, 2Flo, 3Gll
and 4GlO wi~h the fibrinogen:GPIIb/IIIa complex, the
immunoreaotivity of the MAb~ wi~h th~ platelets wa~
oxaminod using platele~ rich pla~ma containing 2-3 mg/ml
fibrinogen.
As ~hown in Table 2, despite the vast
excess of free fibrinogen, each of the four 125I-MAbs
imm~noreacted with fibrinogen:GPIIb/IIIa RIBs on the
:3 ~ t . 1 ~ 5 ~ 13 ~ D :3 S ~;~ ~ e r i e s ~ 1 9--5 ~} ~-- 1 3 8 1~ p . :~ 7
;~000044
- 35 -
platelet surface.
TAB~E 2
125~ gou~
(cpm/plat-let)
Platelets 2G5 2FlO ~ 4GlQ
Non6~imulated 174 662 21~ 678
ADP-stimulated 17,B23 7,471 20,3821~,540
1 0 ~
A6 a further indiaation o~ specificitY of the fsur
depo~ited Mabs for RI~S, ~imilar Mab binding 6tudies were
conduoted u~ing aells containg a Ma~-l receptor rather
than platelets having the GPII~IIIa platelet
glycoprotein receptor. Both Mac-l and GPIIb/IIIa bind
specifically to fibrinogen in a receptor-ligand fashion,
but ~he ~wo intera~ions produce digtinct bi~logical
result~. In the binding 6~dies, the four deposited RI~S
do not exhi~it immunoreaction with fibrinogen-Mac-l
complex, bu~ do immunoreact with ~ibrinogen in a
fibrinogen-GpIIb/IIIa complex. Therefore, the foux Mabs
tested i~munoreact ~peoifically with RlBS on fibrinogen
expreo~ed inaomplex with GPIIb~IIIa but not with RI~S on
fibrinogen when complexed wit~ Mac-1.
3.
Antibodies
T~o hundred ul of isola~ed platelet6
prepared a~ de~cribed in Example 2 were a~mixed with lso
ul Tyrode's buffer ~ontaing ~SA and dextrose ~each at 1
mg/ml), fibrinogen (lmM~, calcium ($mM), and a Fab
fragment of Mab 2F10, prepared ln Example 2 and present
in varying amoUn~s a~ indicated in Table 3. Ten ul of
A~P (80 uM in Tyrode'~ buffer) were then admixed ~o
stimulate platelet aggregation. The admix~ure wa~
maintained at ~ degree~ C whil~ changes in llght
Ic t . 1 ' ~ ~G~S~cM se r i es .S 1~--54~-- 138 ~ P . 3F:
r~nouo4~
- 36 -
transmi~ion of the admlxture w~re monitored over time
using a Dual sample Aggr~gation Meter (Model ~P-247E,
sienco Inc., Morrison, ~0).
The aggregation meter was cal$~rate~ using a
~olution containg 200 ul P~P anfl 200 ul Tyrode's buffer
to ~et a low basaline of light transmisSion at 5 percent
for control aggregations and at 10 percent for
aggregationc in the preeence of antibody. The upper
limit of 100 ml PRP and 300 ul Tyrode's buf~er.
The results o~ained when mea~uring platelet
aggregation inhibition by antibody are ~hown in Table 3,
and are expressed a~ a percen~ of light transmis~ion
(100%) obtained in the ab~ence of an~ibodY when measured
~bout 3 to 4 minu~e~ after ADP wae admixed.
AB~E 3
INHIBITIO~ OF PLATELET
AGGREGAT~ON BY MONO~L4NAL ANTIBODY 2F10
Percent
~FAB] Transmis~ion
o uM 100
O.25 u~ 79
0.5 UM 62.5
1.25 UM 34.5
251.87 uM 17.5
The re~ults in Table 3 show that ~he Mab 2F10
fragment produced a dose-dependent inhibition of platelet
aggreqa~ion. ~hus, the re~ulta indicate the effective
doeages useful to inhibit platelet aggregation and
proce~e~ involving platelet aggregation, such ae
thrombus formation, when using an~ibodies of the present
invention that immunoreact with a RI8S 6pecific ~or
fibrinogen: GpIIb/IIIa complex.
The foregoing specification, includinq the
specific embodiment, ~ B intend~d to be illustrative of
~c: t . 1 ~ ~5~ 1: 0 1 0 0 0 0 D~355a~ 1 se r i ~c ~ 15~--54~-- 138 0 p . 35~
~nO~044
- 37 -
th~ present invention and is not to be taken as limiting.
Numerous other variatiOns and modifica~lon~ can be
effected without departing from the true spirit and ~cope
of the novel conaept~ of this invention.
