Note: Descriptions are shown in the official language in which they were submitted.
WO 93/~Og34- PCI/US~2/05500
211081~
TISSUlE: SPECIFIC IMAGING AGENTS
IJSING INT~L IMAGE ANTI-IDIOTYPIC ANTIBODIES
Field of ~he Inventi~n
The pres~nt invention relat~s generally to the use o~
labeled antî-idiotypic antibodies for diagnostic imaging
and, more particularly, to labeled anti-idiotypic
antibodies ~or use as agents to image in ivo receptors in
biological systems for diagnostic use.
: :Ba~qround ~f the Invention
,~", :
lOThe idiotypic network theory of Jerne (Jerne, N.K.,
~: Anals. ~nst.: Pasteur Paris 125c, 372) proposeæ that the
variable regions of angibodies ~ i o ~ - idiotypes) act ~s
immunogens to give rise to a secondary s~t oX antib~di~s
:cal~led "anti-$diotypes". In particular, if antibodies are
: 15 deve1oped against a ligand that binds`to a cer~ain receptor
wit~in ~he ~body, then the resulting anti-idiotypic
population may contain antibodies that will likewise bind
to~the same~receptor due: to each the ligand and the anti-
idiotypic antibody having similar topological features.
2~:~ Essentially;the:anti-idiotypic antibody mimics the lisand.
Application:o~ the principles proposed by ~ern~ has led to
the~development:of a ~nu~ber of anti-idiotypic antib~die~
such::as those against acetylcholine, TSH, glu¢ocor~icoid,
adenosine and similar such compounds, without ever having
to isolate~and purify tha natural receptor (Erlan~er, B.F.,
: Inter. Re~O Immunol., 59 198gr 131) which can be quite
dif~f ic~lllt .
The use of radiographic imaging agents for the
:viæualiæa~ion of skel~al structures, organs, or tiss~es is
also: well known ~in the area of biological and medical
~ :: :research and diagnostic procedures. The proc~dure whereby
:
W093/0~34 PCT/~S92/05500
~ ~ Q ?~
~- 2
such imaging is accomplished generally involves the
preparation of radioactive agents, which, when introduced
to the biological subject, are localized in the specific
skeletal structures, organs, or tissues to be studied. The
localized radioactive agents may then be traced, plotted,
or scintiphotographed by radiation detectors such as
traversing scanners or scintillation cameras. The
distribution and relative intensity of the detected
radiation indicates the position of the agent in the tissue
and also show~ the presence o~ aberrations, pathological
conditions and the like. The density and distribution of
the receptors being so imaged, depends on the pathological
state of that particular tissue~
The speci~ic targe*ing of effector molecules to a
particular tissue, such as a tumor, using monoclonal
antibodies is:also well known in the axt ~Halpern, SoE~ ~ et
al ., Di~nostic Imaging, 1983 , 40) . Recently, technetium-
sgm or indium-III labeled anti myosin antibodies have bPen
used to i~age myocardial infarction (Dean, R.T., et al., J.
20 Nucl . Me~., 1989, 30, 934). Each of these approaches to
-imaginq p~rtiGular tissue areas are based upon the ability
of a particular type of cell tv secret~ a particular
substanc~ in a very high concentration compared to other
: :~ cells in the vicinity of the desired area to be imaged.
:: 25 Therefore, a need exists to provide an approach to
site specific internal diagnostic imaging of tissue areas
which does nQ~ necessitate particular cell typ~s having the
ability to secrete a substance in very high concentrations
compared to other cells.
In general, it is an object oP the present
invention to pro~ide an unique receptor ~ediated approach,
as opposed to an approach dependent on the concentration of
W093/OOg34 PCT/US92/05500
2~108~
secretions from various cells as described above, to image
site specific areas of tissue. The particular anti-
idiotypic antibodies of the present invention proYide many
advantages when used as diagnostic agent5 to provide a
means of imaging biological receptors without having to
isolat~ and puri~y the natural receptor.
Additional objects and features of the present
invention will appear from the following de~cription in
which a preferred embodiment is described in detall.
- ~.
.~Q Summary of the Invention
The present inven~ion employs the use of anti-
idiotypic antibodies for site specific diagnostic imaging
of biological receptors without having to isol~t2 and
puri~y the natural receptor. An anti-idiotypic antibody
lS refers to an antibody raised again5t a first antibody which
specifically binds to the antibody binding site or CDR of
the first antibody. The antibody binding site or CD~ of an
antibody is that portion thereof which specifically binds
to the recognized epitope.
. .
~20 The anti-idiotypic antibodie~ of the present inven~ion
; :~ are made by d~eloping an~ibodies against a first antibody
: ~ that binds spe~ifically to a certain desired ligand
direct~d at the receptor within the body. The resulting
anti~idiotypic antibody binds to the same receptor due to
its topological ~imilarity with the ligand.
The whole, fragmented, recombinant anti-idiotypic
antibody or recombinant fragment may be labeled with a
radionuclide ~uch as~ Tc-g9m using a chelate approach
wherein one of t~e preferred chelates is a multidentate
organic compound with three amide nitrogen atoms and one
W093/00934 PCT/US92/05500
thiolate sulfur atom (N,S chelate) bonded to a metal
radionuclide, 99~C, which is also bonded to one oxygen atom
to form an anti-idiotypic antibody complex. The whole,
fra~mented or recombinant anti-idiotypic antibody or
recombinant ~ragm~nt thereof may likewise be labeled by
fluorination or by complexing with a paramagnetic particle.
Following labeling, the anti-idiotypic antibody
complex is then injected into a warm-blooded animal for
site specific diagnostia imaging of the particular tissue
area desired by means of imaging the labeled receptors
thereof.
Detailed Descri3~is~l-of the Inventi~n
The anti-idiotypic antibody employed in the present
invsntion is may be made according to the well established
hybridoma technology as exemplified by European Patent
Application Number 89103738.4, incorporated herein by
reference.
The novel approach of utilizing the anti idiotypic
antibodies for imaging specific receptors withi~ a desired
tis ue area has:two major advantages over the conventional
: methods d~scribed above. First, it avoids having to use
: purified reeeptors to develop the anti-re~eptor antibodies.
Often it is difficult and sometimes impossible to isolate
pure, stable receptors for this type ~f immunization.
: 25 Se~ondly, the attachment of a small molecule for labeling,
e.g., molecules having~a molecular weight of approximately
l000 of less, to a large anti-idiotypic antibody s~ould not
perturb its receptor binding capability sl~niicantly. In
con~rast~ the alassical bifunçtional approach of attaching
metal complexes directly to small effector substances, surh
WOg3/~0934 PCT/US92/O~S00
8 1 ~-~
as drugs or hormones for example, essentially blocks the
re~eptor binding capabilities.
Diagnostically or therapeutically useful radi~nuclide
elements which may be used to label the anti-idiotypic
antibody include technetium, indium, rhenium, yttrium,
gadolinium, gallium, bismuth, fluorine, iodine and the like
which can be coupled to the whole, fragm2nted or
~ recombinant anti-idiotypic antibo~ies or recombinant
; ~ fra~ment~ thereof by any one of th~ sev~ral methods known
in the art. Example me~hods that may be used in the
present inventi~n are disclosed in European Patent
Application assigned publication number 0 284 071 and U.S.
Patent Nu~b~rs 4,659,839; 4,732,974; 4,837,003 and
4,965,392 t eaGh incorpor~ted herein by refer~a~, ~s
di~closed in thes~ referenced patent5, either th~ whole,
fragme~t~d or recombinant anti-idiotypic anti~ody or
recombinant fra9ments thereof can be labeled with
radionuclide chelates in a non-selective manner wherein the
chelate is either ~ound at any location on the anti-
2~ idiotypic antibody or by a 5ite-selective technique. In
: the site-sel~ctive techni~ue, the radionuclide chelate is
:
bound distally from the receptor binding site of the anti-
idiotypic antibody by using, for example, a bifunctional
coupling ag~nt which reacts with a free sulfhydryl group
25~ generàlly found:~in the fragmented or anti-idiotypic
antibody~and is used to~ label the target biologi~al
receptors. A standard method for preparin~ anti-idiotypic
antibody fra ~ ents i~ by the enzymatic digestion of the
: ~ whole antibody wi~h papaln or pepsin as described by
~ 30 Par~am, et al., J. ImEunol. Methods, 19~2, 53, 133. The
:~ anti-idiotyp~c antibody~or fragments thereof can li~ewise
be iodinated dir~c~ly wit~ a sodium iodîde~chlora~ine-T
procedu~e or can be attached via covalently bound
bifunctional moieties suh as those illustrated in Formula
'
: -
`~:
W~93/~Og34 PCT/US92/0550
~ t~ 3 6
I below:
~X
FORMULA ~ :
wherein X is sslected from the group consisting ofisocyanate ~ i sothiocyanate, imid~te, maleimido,
succini~idyloxycarbonyl, acid chloride and sulfonyl
chloride. Fluorines, which are potentially useful for
fluorine magnetic resonance ima~ing lMRI) or for positron
emission tomography (PET) can likewise, for example, be
conjugated to the antibody via a bifunctional molecule as
illustrated ~y Formu~a II below:
~ ..
~J
r
CF3
FORMULA II
wherein Y is defined to be the sam~ as X above.
:Metal ions such as technetium t rhenium, indium,
yttrium, gadolinium, ~ismuth and th~ like can be joined to
either the whole, fragmented or recombinant anti-idiotypic
antibody ~r recombinant fragments thereof in a selecti~e
manner using a bifunctional molecule that contains an
appropriate liga~d and a coupling group that reacts
specifically with th~ protein sulPhydryl groups such as a
maleimido group as illustrated in Scheme I belowD
Alternatively, a non-selective manner may be utilized ~:
~.
W093/0~934 . PC~/U$92/05500
~08:~
wherein the bifunctional molecule contains the ligand and
a coupling moiety selected from the group co~sisting of
succinimidyloxycarbonyl, isocyanate, imidate,
isothiocyanate, acid chloride and sulfonyl chloride such as
illustrated in Scheme 2 below. The ~aleimido ligand 3, the
succinimido ligand 6, and the method of labeling the
conjugated proteins 4 and 7 with indium-lll or technetium-
99m have been described in detail by Nicolotti, et al.,
: U.S. Patent No. 4,732,974, incorporated herein by
lO reference.
'
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W0 93/0~934 P~r/US92/05500
9~
Scheme 1
~N--(CH~ ~N--(CH~
3 ~02H Anti~ 4 ~02H
InC13
An!ibody--SH t
[ I ~ 1
Antibody ~2-
W0 93/00934 P~/U~92~05~0
2:~.L~81i~
Scheme 2
~ , ~ .,
O~NH HN~ O~ ~NH HN~
~S I IN~O O SHN~O
~, Ph~O ~ SOlNa HN'Antibody
N2g9mTco4
An~ibodY--NH2 S~ccharic acid, Sn2
.,
~,~0
~T~ ~
~S N~O
A ~Y
~093/00g34 PCT/~S92/05500
3~
10
The present invention is ther~fore not restricted to
radiographic imaging, and may be applied to any imaging
modality. For example, the anti-idiotypic antibody of the
present invention may b~ likewise labeled through
fluorination or by labeling with a paramagnetic metal
chelate. For further example, the antibody conjugate in
Example 4 below can complex gadolinium or europium for MRI
or immunofluorescence applications respectively.
In a preferred embodiment, the internal image
antibody, i.e., anti-idiotypic antibody i5 directed at the
.~ digoxin receptor in the myocardium and is labeled with
technetium-9gm. It is believed that the labeled digoxin
internal image antibodies may be useful in the dia~nosis of
some coronary disorders and may supplement the information
gained ~rom:the use of myocardial perfusion agents such aæ
thallium-201~
he novel imaging agents of this invention ~.an be
ormulat~d into; diagnostic compositions containing
suf~icient amount~of labeled anti-idiotypic antibody ~or
20: imaging,: to~ther with a pha~maceutiaally acceptabla buffer
suc~ as phosphate, citrate, or tris~hydroxymethyl~amino-
methane; balanced ionic solutions c~n~aining chloride and
bicar ~ nat~ salt~ of ~lood plasma cations suGh as Ca2- 0 N~,
: K-,~Mg~, saline and~the like.
The concentration;of the imaging aqent accordin~ to
the present in~ention sh~uld b~ sufficient to provide
sa~isP~c~ory imaging, ~ . a . 1 to 5û mil l icuries . Jrhe
: ~ imaging a~ent should be administered so as to remain in the
atient for 1 to 3 hours, although both longer and shorter
~: 30 time periods are acceptable. Therefore, convenient ampules
containinq 1 to 10 mL of aqueous solution~ may be prepared.
.~
W~93/00934 PCT/US~/05500
Imaging may ~e carried out in the normal manner, for
example by in~ecting a sufficient amount of the imaging
~omposition to provide adequate imaging and then scanning
with a suitable machine, such as a gamma camera.
.
The anti-idio~ypic antibodies and the correspondinq
radionuclide conjugates can be prepared in accordance with
the examples set forth below, which are not intended to be
limiting.
EXAMPLE 1
;J,I~ Fusion c~f mouse myeloma cells with th~ sple~n cells of ALJ
mice im~unized with Balb-C mvuse a~ti-digoxin antibody.
~onoclonal antibodies were produced by the hybridoma
technology well known in the art. Two AJ mice were
~ i~munized wi~h murine (Balb-C) monoclonal anti-digoxin
antibody (Medex Laboratories). A booster injection was
: : giv~n 3 weeks after the primary immunization and the
spl~ens wer~ removed a~te~ 3 days. ~ouse myeloma and the
~ ~ ~ spleen cells were washed three ti~es wi~h Dulbeccc's ~agle
:~ ~ Medium ~ME) and suspended in DME (lOml). A ~ mL portion
o~ each of these cell suspensions were mixed and
~ centri~uged. The supernatant was dîscarded and the pellet
: ~ ~w~s treated with l m~ of polyethylene glycol ~added over a
45 second period),: 3 mL of DME ~added over 30 second
period~), and additional 9 mL of DME added over a 30 second
period. The cells were allowed to stand at ambient
temp~rature for 8 minu~es and at 37 C for 2 minutes. The
cells were centrifuged~ suspended in HAT medium (lO mL1, :
~: and distributed in microtit~r plates. The cells were
allowed to grow and wers screenad by radioimmunoassay
procedure approximately 3 weeks after fusion.
:
~:
WO 93/00934 - PC[/US~2~V~5~0
12
EXAMPLE 2
Screeni~g of anti-idio~ypic antibodies f or digoxin .
A 75~L portion of affinity purified goat anti-mouse
antibody ( 2 mg/mL) was dilutsd with PBS buffer ( 150 mL~ .
5 In each well was placed 200 ~L of the a~ove antibody and
the plates wers incubated at 37 C for 4 hours. The plates
were washed with water ( 3 times ~, treated with 3~ BSA
solution ( 200 ,uL), and incubated for 1 hour. The wells
w~re washed again with water ( 3 times ) and then treated
10 with the super~atant~ from the cell . culture ~150 ~L) and
,~, allowed to incubat~ at ambient temperature for about 18
hours . The plates were washed with water ( 3 times ),
treated with '25I labeled goat anti-digoxin ( lO0 ~L) and
incubated for 4 hours., Thereafter, the plates wer~ washed
15 and the wells were counlted. A total of 39 pc~ ive well~
wer~ idelltified.
The supernatants from the positive wells above ( lûO
~L) were mixed with l25I-digoxin ~ 50 ~L) and were placed in
the microtiter plates which wer~ preYiously coated with
20 approximately Q . 5 ,ug of ~onoclonal mouse anti-digoxin
anti~dy îor 1 hour. Th~ plates were washed with water 3
times and counted. Inhibition of ~a5I-digoxin, compared ~o
the control, indicated a positive test f or anti-idiotypes .
F~lar~;?Q~i~iv~ wells were identified.
2 5 EXAMPLE 3
Preparation of F t" fragmen~ of anti-idiot~pic digoxin
anti~dy .
Ascites f luid is obtair~ed in the usual ~Qann~r by the
ir~ jectioal of the hy~ridoma cells from Exampl2 2 into mouse
perits:)nell~ . It i~ purif ied by three succ:essive
precipitation with anmoni-lIG sulfate usiny 20mM phosphate
.
W093/OOg34 PCT~US92/0~500
buffer, pH 6.8. Thereafter, the protein is dialyzed
exhaustively using 20 mM phosphate buf~er, pH 6.8. The
monoclonal antibody is purified by ion-exchange
chromatography (Whatman C-52 column, 0 to 500 mM NaCl
gradient in pH 6.8 phosphate buffer). The desired fraction
is collècted and stored in the same buffer at 4 C.
~:~ :The desired amount of antibody (absorbance of 1~
solution at 280 nm is 14.4) and cysteine-free papain
(Worthington, 2 times crystallized) in the ratio of 1:20
are incubated at 37~C using 10 times the volume of
digestion: buf~er (100 mM sodium acetate, 3 mM disodium
EDTA, pH 5,5) until the reaction is complete (3-16 hours)
a~ determined:~by~ SDS-PAGE The digestion mixture is
diafiltered tAmicon~low c~ PM-10 membrane~ at 4 C using
15~ TRIS buffer,~pH 7.2~. It~is then applied to the Whatman DE-
52 ion-exchange resin, previously equilibrated in the same
buff:er, to re~ove the anionic Fc fra~ment. ~he eluent,
which~consists of ~ (F~,), and inactivated papain, is purified
by~Sephadex G-lOO`siz;e~exclusion chromatography using TRIS
:20~ buffer,;pH 7.2.~ The ~desired antibody fragment elutes i~
the~void vol;ume~ and is characterized by SDS-PAGE. It is
stored as frozen aliquots~at -70-Co
, :..
: m~ :dimer~;~thus obtained by papain diges~ion of the
whole~ antibody ~is then:fu~ther cleaved to the desired F D~
:25~ ~fragment ~u~Sing~ thiol ;reagents Buch as cysteine or
dithiothreitol.~ :The~dimer in 25 mM phosphate buffer, pH
7.4, containing 2 mM disodiu~ ~DTA and 0.02% (w~v) sodium
azide is:incubated:at:room te~perature with either cysteine
~or~ dithiothreitol until~ the reaction is complete as
; 30: determined: by SDS-PAGE (usually 1-4 hours). Excess
~ reducing agent:and other low molecular weight ~ragments are
:
quickly removed by Sephadex G-25 column usi~g PBS. The ~D
fragment thus obtalred should be used as soon as possible
..
.
W~93/00934 PCT/US92J05500
b~
14
in order to pre~ent the oxidation of the sulfhydryl groups.
EXAMPLE 4
Site-selective labeling of F~b, fragments with indium-lll.
A mixture of the F~b~ fragment and about 20 fold excess
of the ligand shown in Scheme 1 is incubated in labeling
buffer (50 mM MES, pH 6.0) for 2-4 hour~. Excess ligand
and other low molecular weight impurities are quickly
r~moved by Sephadex G-25 column using the lab~ling buffer.
~~ The antibody ~ragment co~jugated with the ligand is
then labeled with radioactive indium chloride as described
below. A mixture of ~InCl~ ~80 ~L) and 4,5-dihydrQxy-1,3-
benzenedisulfonic acid (40 ~L, 10 mM) in 0.2 M ~ES buffer
; (80~L) is treated with the conjugated F~, fragment and ~he
entire mixture is incuba~ed at room temperature Por 1 hour.
The reaction mixture is treated with 0.2 M EDTA ~40 ~L~ to
remove excess indium. The indium labeled antibody is then
: ~
purified by Sephadex G-50 column using 0.15 M NaCl as :
eluent.
~ .
EXAMPL~ 5
20 No~-site-selective labeling of F.b, fragments with
~ techn~tiu~-99m.
:
, -
` A mixture of the F~, fra~ment and about 25 fold excess
of the ligand shown in Scheme 2 i~ incubated in 25 mM
phosp~ate bu~fer, pH 7.4 at room temperature for about 30
minute~. Excess ligand and other low molecular weight
: impur~ities are quickly removed ~y Sephadex G-25 column
: usi~g the same buffer. Therea~ter, the conjugated F~,
:: solution ~as treated with 15 ~L 9g~Tc-saccharic acid and themixture is incubated at 37 C for about 3n minutes. The
technetium labeled antibody is then purified ~y Sephadex G-
W0~3/00934 - PCT/US92/0~00
2 1 .i 3 ~ 1 9
25 column using the same buffer.
In order to image biological receptors, a preparation
of the present in~ention using either whole, fragmented, or
recombinant anti-idiotypic antibodies or a recombinant
5 fragment thereof îs administered to the patient, for
example, in the form of an in jectabl~ liquid. By means of
suitable detectors~ e.g., a gamma camera, images can be
obtained by recording the emitted radiation of the organ or
.
the pathological process in which the labeled anti-
~lo idiotypic antibody has been incorporated, which in the
: present case i5 biological receptors.
:
The anti-idiotypic antibody of th~ present invention ~:
or a fra~ment or~recombinant derivative thereof prepared as :
described a~ove provides a means o~ Ln Y~Q diagnostic ~;
: lS imaging of receptors which provides many advantages over ~:
prior known procedures: which involve cellular s~cretions.
: ~ :
After the anti-idiotypic antibody is pr~pared and : :
labeled acr~rding to one of the procedures described, the
oompos~ition is~ used with a pharmaceutically acceptable
0~ ca~rier in a: method o~ performing a diagnostic i~aging :~
procedure using a :gamma camera or like device which
involves injeoting or ~administering to a warm-blooded
animal an:effectiv~ aDount of the present invention and
: then exposing the warm-blooded animal to an imaging ::~
25 pxocedure as des~ribed above, thereby imaging at least a ~:-
portion of the~body of the warm-blooded animal. -:~
Pharmaceu~ically ~acceptable carriers in~lude those ~:
that ar~ suitable for: injection such as aqueous buffer
solutions, e.g., tris(hydroxymethyl)aminomethane (and its :~
3~ salts), phosphate, citrate, bicarbonate, etc~, ~terile
:: : :
WO ~3/00934 PCI /l 1S92/~)5500
16
water for injection, physiological saline, and balanced
ionic solutions containing chloride and/or bicarbonate
salts of normal blood plasma cations such as Caa~, Na', K~
and Mg2~. Other b~ff~r solutions are described in
5 Remin~ton's Practice of Pharmacy, Eleventh Edition, for
example on page 170. The carriers may contain a chelating
agent, e.g., a small amoun~ of ethylenediaminetetraacetic
acid, calcium disodium salt, or other phar~aceutically
acceptable chelating agents.
,
The concentration of the labeled anti-idiotypic
~-~ antibodies in the pharmaceutical~y acceptable carrier, for
example an aqueous medium, varies with the particular field
of use. A sufficient amount is present in the
pharmaceutically acceptable carrier in this particular case
when satisfactory vi~ualization of the receptoræ is
achievable.
~: The compositi~n is administ~red to the warm-blooded
animal so that the composition remains in the living animal
body for about 6 to 7 hours, although shorter and longer
residence periods are normally acceptable~
: The labeled anti-idiotypic antibodies may be used in
the usual way in im~ging procedures. For exa~pl~, with the
~ : :present invention :when imaging bio~ogical rec~ptors, a
: ~sufficient amount of the labeled anti-idio~ypic antibody
: 25 must be intravenously administered t~ the warm-blooded
animal to provide adequate visualization; the animal or a
portion thereof is then scanned with a suitable imaging
machine such as a ga~ma amera.
After co~sideration of the above specification, i~
will be appreciated that many improvements and
modifieations iD the details may be mad~ withou departing
WO g3100934 Pcr/uss2/ossoo
from the spixit and scope of the inve~tion. It is to be
understood, therefore, that the invention is in no way
limited, except as defined by the appended claims.
;
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