Note: Descriptions are shown in the official language in which they were submitted.
WO 91/14458 PCl`/US91/01637
2 ~ 7 .~
A F~NCTION~LI 2:E:D COMPLE~AND
BACKGROUND OF THE INVENTION
Technical Fleld
The present invention relates to macrocyclic
chelates and methods of use thereof. Specifically, the
invention relates to a substituted l,4,7,10-
tetraazacyclododecane triacetic acid wherein the three
triacetic acids are linked at three of the nitrogens of
the macrocycle, and a hydrogen is attached to the
fourth nitrogen of the macrocycle. The chelate exists
as a mixture of the four possible structures (or
isomers). The present invention also relates to the
various uses of these particular chelating agents.
Background Information
Macrocycles have been studied for their
usafulness as chelating agents for numerous metal ions
that have therapeutic, diagnostic, or other uses. A
macrocycle of considerable value as a chelate is the
1,4,7,10-tetraazacyclododecane-N, N', N'', N'''-
tetraacetic acid (DOTA). ~OTA type compounds have been
linked to biomolecules to form delivery systems for the
chelated metal ion to specific sites in vivo.
U.S. Patent 4,678,667 to Meares et al.,
discloses such chelating agents. The chelating agents
of this patent can include a bifunctional DOTA compound
that is a Cu(II) chelate. The usefulness is therefore
limited to the effects of the copper metal ion. The
synthesis reportedly also lacks in efficiency resulting
in low yields and not always reproducible results.
U.S. Patent 4,622,420 to Meares et al.
discloses acyclic bifunctional chelating agents of the
ethylenediamine N, N, N', N'-tetraacetic acid (EDTA)
.
.
wo9l/l~s~ PCT/US91/01637
2~7~2~
types useful for binding metals, other than copper,
such as Indium. These compounds are useful for imaging
tumors.
U.S. Patent 4,652,519 to Warshawsky et al.
also discloses EDTA type bifunctional chelates and a
process for their production. These compounds were
offered as an alternative to the Meares compounds
discussed above.
U.S. Patents 4,453,106 and 4,474,509 to Gansow
et al. disclose the use of metal chelate conjugated
monoclonal antibodies and specific metal chelate
conjugated monoclonal antibodies, respectively. These
disclosures provide compounds and methods for treating
cellular disorders. Advantages of the use of chimeric
and monoclonal antibodies have been discussed
(Morrison, S.L., Hospital Practice (Office Edition)
24:64-65, 72-74, 77-80 (19893. Radiometal chelate
conjugated monoclo~al antibodies specific to a target
cell are used to deliver alpha, beta, or Auger electon
emitting metal ion. These disclosures are not related
to DOTA compounds.
European Patent Application 0 088 695 to
McKearn et al. discloses the oxidation of monoclonal
antibody and linking of ligand by imine formation and
subsequent cyanoborohydride reduction to form
conjugates.
PCT application (WO 89/01476) to Parker et al.
discloses macrocycle ligand/chelates and conjugates
thereof.
European Patent Application 0 292 689 to
Tweedle et al. discloses triacetate 1,4,7 10-tetraaza
macrocycles but does not discuss the linkage of these
WO91/1~58 PCT/US91/01637
2~7~2~
compounds to haptens.
Other U.S. patents which disclose various
chelating agents or conjugates include U.S.P.
4,442,305, U.S.P. 4,S30,963, U.S.P. 4,732,974, and
U.S.P. 4,585,559.
The value of having a ligand conjugate to
chelate metal ions of therapeutic, diagnostic, or other
uses lies in their commercial importance. This
commercial importance is created by the fact that many
metal ions have desirable characteristics, but the
delivery systems lack specificity to target the metal
ions, or do not adequately bind the metal ions.
Examples of the usefulness of specific metal ions
follow.
The usefulness of radionuclide materials in
cancer therapy is disclosed in the article, Kozak e~
al., "Radionuclide-Conjugated Monoclonal Antibodies: A
Synthesis of Immunology, in Organic Synthesis and
Nuclear Science," Trends in Biotechnolooy 4(10): 259-
264 (1985). This article discusses the use of antibody
- conjugates to deliver either alpha or beta radiation.
Other uses for chelated metal ions are
- discussed in Magerstadt et al. "Gd(DOTA): An
Alternative to Gd(DTPA) as a T1/2 Relaxation Agent for
NM~ Imaging or Spectroscopy," Maanetic Resonance in
Medicine 3:808-812 (i986). Specifically, this article
discloses the usefulness of gadolinium as a relaxation
agent for NMR imaging.
The efficacy of a linking group, within
- 30 chelated metal complexes, has been discussed by Paik et
~ al., J. Nucl. Med. 30:1693~1701 (1989).
.
.' ' ' .
WO9]/1~58 PCT/US~1/01637
2~732~
Other articles which may be of some interest
include- ~cMurry et al., "Template and Stepwise
Synthesis of a Macrobicyclic Catechoylamide Ferric Ion
Sequestering Agent," J. Amer. Chem. Soc. 109: 3451-53
(1987), Raymond et al., "Macrocyclic Catechol
Containing Ligands," Pure & A~Pl. Chem. 60: 545-48
(1988), ~cMurry et al., "Macrobicyclic Ion (III)
Sequestering Agents," J. Am. Chem~ Soc. 109: 7196-98
(1987), McMurry et al., "Molecular Recognition and
Metal Ion Template Synthesis," Science 244: 938-43
- (1989), Kiggin et al., "Functionalized Oligocyclic
Large Cavities- A Novel Siderophore," Anqew. Chem. Int.
Ed. Enql. 23: 714-15 (1984), Kiggin et al., "Large
Oligocyclic Cavities for Strong Cation Complexation,
Tetrahedron 42: 1859-72 (1986), Sun et al., "New
Multidentate Ligands. 28. Synthesis and Evaluation of
New Macrocyclic Ligands Containing Bidentate Endocyclic
Catechol Donor Groups," Inora. Chem. 25: 4780-85
(1985), Moi et al., "The Peptide Way to Macrocyclic
- 20 Bifunctional Chelating Agents: Synthesis of 2-(p-
Nitrobenzyl) -1,4,7,10- Tetraazacyclododecane -N, N',
N ", N' " -Tetraacetic Acid and Study of Its Yttrium
(III) Complex, " J. Am. Chem. Soc. 110:6266-67 (1988),
and Cox et al., "Synthesis of a Kinetically Stable
Yttrium-90 Labelled Macrocycle Antibody Conjugate," J.
Chem. Soc., Chem. Commun., pp. 797-98 (1989).
All patents and publications referred to
herein are hereby incorporated by reference.
The industry is lacking a macrocyclic chelate
that can be efficiently produced in high yields, that
can be linked to proteins, and that has desirable
chelating qualities for numerous metal ions wherein the
WO91/1~58 P~T/US9l/01637
2~7~
formation kinetics thereof and reaction conditions
required for metal complexation are favorable for use
with monoclonal antibodies.
SUMMARY OF THE INVENTION
The invention is the ligand and its metal chelates
having a general Formula of I and Ia, respectively:
~ n ~) rl
I I o.
wherein three of the R groups are -CH2COOH with the
fourth ~ being kept as -H;
n is an integer from 1 to 5;
X equals -NO2 or -NH2; and
20 and M is a metal ion being a member selected from the
group of elements consisting of Y, In, Bi, Pb, Cu, Ag,
Au, Pt, and the Lanthanides.
A preferred embodiment includes the situation
where, in the ligand, X is either -NO2 or -NH2, and n
is an integer from 1 to 2.
Another preferred embodiment include- the
situation where, in the chelate, X is either iO2 or -
NH2, and n is 1 to 2.
An additional embodiment includes the
situation where, in the chelate described in the
paragraph directly above, M is a member selected from
the group consisting of Bi, Pb, Y, Cu, Gd, Eu and Tb.
~. .
W~91tl~58 PCT/US91/01637
2 ~ 9 2 ~ ~ 6
A further embodiment includes the situation
where, in the chelate, n is 2, X is -NH2, and M is a
member selected from the group consisting of Pb203,
pb2l2 Bi2l2 y90, and Cu67.
An additional embodiment includes the chelate
described in the paragraph directly above wherein M is
a member selected from the group consisting of Pb2l2
and Bi
Another embodiment includes the situation
where, in the chelate, n is 2, X is -NH2, and X is
-NH2, and M is a member selected from the group
consisting of Eu and Tb.
The invention also includes ligand-hapten
conjugates of the general Formula II and chelate-hapten
conjugates of the general Formula IIa:
~ X~
~ IIo_
wherein three of the R groups are -CH2COOH with the
fourth R group being kept as -H;
n is an integer from l to 5;
X' equals -NH-Q where Q is selected from the group
consisting of hormones, steroids, enzymes and proteins,
a subset of proteins being monoclonal antibodies,
chimeric antibodies, and fragments thereof;
. . .
WO9l/1~58 PCT/US91/01637
2~92~
and M is a metal ion being a member selected from the
group consisting of Y, In, Bi, Pb, Cu, Ag, Au, Pt, and
the Lanthanides.
A further embodiment includes the ligand-
hapten conjugate wherein n is 2 and X' is-NH-Q.
Another embodiment includes the ligand hapten-
conjugate wherein Q is a protain, said protein being a
monoclonal antibody, chimeric antibody, or fragments
thereof.
A further embodiment of this invention is a
ligand-hapten conjugate, as is drawn in Formula II
(shown above), wherein three groups are -CH2COOH, and
the fourth is -H, n is an integer from 1 to 5, and X'
is -NH-L-Q where Q is a hapten chosen from the group
consisting of hormones, steroids, enzymes, proteins,
monoclonal antibodies, chimeric antibodies, or
fragments thereof, and L is a covalent linking group.
An additional embodiment of this invention is
a chelate-hapten conjugate, as is drawn in Formula IIa
(shown above), wherein three groups are
-CH2COOH, and the fourth is -H, n i~ an integer from 1
to 5, X' is -NH-L-Q where Q is a hapten selected from
the group consisting of hormones, steroids, enzymes,
proteins, monoclonal antibodies, chimeric antibodies,
or fragments thereof, M is a metal ion selected from
the group consisting of Bi, Pb, Y, Cu, Ag, Au, Pt and
the lanthanides, and L is a covalent linking group.
In either of the two embodiements directly
above, L may be an organic radical or a substituted
aliphatic hyrdocarbon chain, optionally interrupted by
one or more hetero atoms selected from -0- or
.
WO91/1~5~ PCT/US9t/01637
2~792~
-S-, or by one or more -NR'- groups (where R' is a
hydrogen atom or a Cl C alkyl group), -CONR'- groups, -
NR'CO- groups, cycloaliphatic groups, aromatic groups,
or heteroaromatic groups, or a mixture thereof.
The invention also includes methods for using
these compounds for treatment of cellular disorders and
for diagnostic tests. Thus, the above compounds may be
used as therapeutic and diagnostic agents.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 illustrates the chemical pathway used
to produce the preferred embodiment of the invention.
DETAILED DESCRIPTION OF THE INVENTION
Compounds of this invention include the
substituted 1,4,7,10 tetraazacyclododecane triacetic
acid represented in the general Formula I shown above
or specifically by compound 5 of Figure l. The general
formula is a 12 membered ring tetraaza macrocycle, with
the nitrogens in the 1,4,7,10 positions. The acetic
acid groups are attached to three of the nitrogens, and
a hydrogen is attached to the remaining secondary
nitrogen. This description encompasses all four
possible structures or isomers contained in the
mixture. Furthermore, each of the nitrogens is bridged
by an ethylene group.
The substituted triacetic acid ligands
represented by Formula I complexes metals (Formula Ia).
Metal complexes are formed by placing the ligand into
solution with an appropriate metal salt having the
metal to be chelated. Metal salts have to be selected
so as to prevent the hydrolysis of the metal. Also,
reaction conditions in an aqueous medium have to be
chosen such that the metal is not hydrolyzed. For
WO91/1~58 PCTIUS91'01637
2 i~ 7.~
example, a lead nitrate, bismuth iodide complex, or
yttrium acetate salts can be used to form a metal
chelate with lead, bismuth, or yttrium, respectively.
General examples of suitable salts include any soluble
divalent metal complex or any trivalent metal complex
that is not hydrolyzed at pH 4 to about 9. The most
desirable metal ions for chelation with general Formula
I are members from the group consisting of bismuth,
lead, copper, yttrium, platinum, gold, silver, gallium,
and any of the elements of the lanthanide series. The
most desirable elements of the lanthanide series are
(1) gadolinium, for use in NMR imaging and as a
relaxation agent in NMR imaging, and (2) terbium and
(3) europium, because of their use as chromophores in
time resolved fluorescence spectroscopy. These
fluorescent compounds can be useful in an in vitro
; diagnostic assay.
The aniline substituent of compound 5 is
desirable as a substituent that can be used to
conjugate the compound to haptens. The aniline group
can be linked to an oxidized carbohydrate on the
protein as an imine, ~nd the linkage can subsequently
be reduced to an amine by cyanoborohydride.
The haptens suitable for linking with the
compounds of general Formula I or Ia can vary widely.
The most desirable haptens are members selected from
the group consisting of hormones, steroids, enzymes,
proteins, monoclonal or chimeric antibodies, and
fragments thereof. These haptens are desirable because
of their site specificity to tumors and/or various
organs of the body. The preferred hapten for use in
treating cellular disorders or various disease
.
WO91/1~58 PCT/US91/01637
~07~2~
conditions is a specific protein type, a monoclonal
antibody or chimeric antibody, or fragments thereof.
In Formulas Ia and IIa, the compounds of this
invention can have a value of n equal to an integer
from 1 to 5. In a preferred embodiment, n equals 2.
It is desirable for n to equal 2 versus 1 because the
chelating ligand is separated form the protein more and
possesses increased free rotation. The increased free
rotation allows a metal to chelate with the
macromolecule more easily. When n is 3 or greater, the
synthesis of the compound becomes lengthy.
Figure l illustrates the preferred reaction
pathway or process for forming the compound of this
invention. The process first provides a cyclic
triamide with a substituent on the carbon backbone
framework of the molecule. The embodiment of Figure 1
has n=l as the initial substituent for linkage. The
process then provides a tetraaza macromolecule having
the substituent in the 2 position as shown. Alkylation
with bromacetic acid forms the three nitrogen to carbon
bonds of the three carboxymethylene substituents.
The desired diactive ester 1 is formed
sequentially from iminodiacetic acid. The amine is
first blocked using the reagent BOC-ON or any other
suitable blocking agent such as EMOC, in the presence
of triethylamine which serves to deprotonate the
starting material. The subsequent nitrogen blocked
diacetic acid or other such nitrogen blocked compound
is then coupled to N-hydroxysuccinimide, or any of the
suitable compounds such as phenols, or N-
hydroxydicarboximides which form a reactive ester. The
choice of compounds which form active esters or
WO91/1~8 PCT/US91/01637
2~7~2~
11
blocking groups in within the scope of the art. The
coupling is done by a carbodiimide. This step produces
the nitrogen blocked active ester.
Ring formation under high dilution conditions
between amino acid amides (compounds la) with the
nitrogen blocked active ester (compound 1) is then
performed. This step forms the triamide macrocycle
(compound 2). The amine nitrogen of compound 2 is
deblocked with hydrochloric acid in dioxane. This
forms the HCl salt of the triamide macrocycle. The
salt is then reduced with borane/tetrahydrofuran. The
resulting borane adduct is cleaved by hydrochloric acid
to form the substituted tetraaza macrocycle (compound
3). This macrocycle can then be alky` ted with three
equivalents of haloacetic acid in the presence of base
to form a nitrobenzyl tetraaza macrocycle triacetate
(compound 4). The nitro group of compound 4 can be
reduced with hydrogen over a palladium on carbon
catalyst to produce the aniline or the aminobenzyl
structure depicted as compound 5.
The chelating agent formed by the tri-
- alkylation of the cyclen described in the process of
Figure l is novel and separate from those previously
described in that, when reacted with trivalent metal
ions, an electrically neutral (zero charged) metal
chelate is formed. The advantages of such zero charged
chelates in medical applications is well understood and
has been recently described, for example, by Tweedle,
et. al. (E.P.A. O 292 698), and includes reduced
osmolality of the complex and increased lipophilicity,
` hence solubility in lipid tissues. This, in turn,
confers an increased ability of the metal complexes to
.
WO91/1~58 PCT/US91/01637
2'`37 92~
clear from the interior of cells. This last feature is
of importance since often monoclonal antibodies are
catabolized into normal liver cell interiors, and for
diagnostic imaging in the abdomen, such clearance from
normal liv~r cells is to be desired.
A particular advantage of the tri-alkylated
ligands of Formula I lies in their ability to rapidly
form metal chelates at aqueous solution pH values 4-7.
The more complex DOTA macrocycles, in which the
hydrogen that is attached to the secondary nitrogen of
Formula I is replaced by a carboxymethylene group, are
harder to label with metals since they react
exceedingly slow with trivalent metals such as indium
or yttrium at these pH values, thus, if a radiometal is
used, providing an undesirable long term radiation does
during metal chelate formation to any hapten to which
the ligand is conjugated. The pH region 4-7 is
especially desirable for formation of trivalent metal
ion chelate conjugated monoclonal antibodies in that
the antibody is stable therein, and the metal ions are
less susceptible to hydrolysis as occurs at higher pH
values, thus markedly simplifying the radiometal
labeling of ligand conjugated antibodies or fragments
as compared to such procedures for hapten conjugated
DOTA ligands.
In its preferred embodiment, the coupling of
compound 5 to antibodies is through imine formation
with oxidized carbohydrate on protein followed by ln
situ reduction to the amine. An advantage of this
methodology is that when coupling to proteins and, in
particular, when coupling to antibodies, the
carbohydrate of the antibody can be oxidized prior to
WOglt1~58 PCT/US9l/01637
2~9~
the coupling reaction. The aniline reacts with the
aldehyde that is formed on the protein. The imine that
i5 formed can be reduced by cyanoborohydride to form a
covalent amine linkage to the antibody that is site
specific. This position is distant from the antigen
binding sites of the monoclonal antibody and thus
minimizes any deleterious effects of the protein
modification.
An embodiment of the invention involves a
ligand-hapten conjugate of general Formula II.
This conjugate complexes metal ions. For complexation,
especially of radioactive metals, it is desirable to
expose metal ions to the protein conjugate in a
concentrated metal solution for a short a period of
time as possible. Certain metals, such as divalent
metal ions, react rapidly and directly with the
conjugate. The kinetics of the formation reactions for
these compounds are so rapid that it is desirable to
have the ligand-hapten conjugate available in the
pharmacy immediately prior to u~e. The conjugate can
then be mixed in the radionuclide to form the complex
and, subsequently, the metal chelate conjugate formed
can be purified, for example, by size exclusion high
pressure liquid chromatography. A desirable hapten for
the ligand conjugate can be selected from the group
consisting of hormones, steroids, enzymes, and
proteins.
The most commercially useful embodiments of
the invention are metal chelate-hapten conjugates
having the general Formula IIa where: (1) n is an
integer from 1 to 5, (2) X is -NH-Q- with Q being a
hapten selected from the group consisting of hormones,
WO91/1~58 PCT/US91/01637
2 ~ 7 ~ 14
steroids, proteins, monoclonal antibodies, chimeric
antibodies, and fragments thereof, and (3) M is a metal
ion being selected from the group of elements
consisting of Bi, Pb, Cu, Ag, Au, Y, Pt, and the
5 lanthanides. These chelate conjugates can deliver
radioactive metal ions such as Pb212, Bi212, Y90, and
Cu67 to treat specific cellular disorders. A
further embodiment of this invention is a ligand-hapten
conjugate, as is drawn in Formula II (shown above),
wherein three groups are -CH2COOH, and the fourth is -
H, n is an integer from l to 5, and X' is -NH-L-Q where
Q is a hapten chosen from the group consisting of
hormones, steroids, enzymes, proteins, monoclonal
antibodies, chimeric antibodies, or fragments thPre and
L is a covalent linking group.
An additional embodiment of this invention is
- a chelate-hapten conjugate as is drawn in Formula IIa
(shown above) wherein three groups are -CH2COOH, and
the fourth is -H, n is an integer from l to 5, X' is -
NH-L-Q where Q is a hapten selected from the group
consisting of hormones, steroids, enzymes, proteins,
monoclonal antibodies, chimeric antibodies, or
fragments thereof, M is a metal ion selected from the
group consisting of Bi, Pb, Y, Cu, Ag, Au, Pt and the
lanthanides, and L is a covalent linking group.
In either of the two embodiments directly
above, L may be an organic radical or a substituted
aliphatic hyrdocarbon chain, optionally interrupted by
one or more hetero atoms selected from -0- or
-S-, or by one or more -NR'- groups (where R' is a
hydrogen atom or a Cl C alkyl group), -CONR'- groups, -
NR'CO- groups, cycloaliphatic groups, aromatic groups,
WO91/1~8 PCT/~S91/01637
2~9.~
or heteroaromatic groups, or a mixture thereof.
The invention includes a process for treating
cellular disorders. The process uses the chelate
conjugate with a hapten having a selective binding site
at the cellular disorder. For example, ~ can be a
monoclonal antibody wherein the antibody is created and
directed against an epitope found specifically on the
tumor cells. Thus, when Y90
is transported to the antigen site and decays, a beta
irradiation is produced. If desired, Bi2l2 can be
introduced in those cases where the disorder to be
treated, such as with leukemic cells, can be reached
within the l hour half-life of the isotope. Most
desirably, at least 95 percent of the radionuclide
remains in the chelate. In an acidic medium, such as
the stomach, at least about 70 percent is retained.
The invention also includes a process for
diagnostic testing. This process uses a chelate
conjugate having general Formula IIa wherein M is
selected from the group consisting of Pb203, Inlll,
- Ga67, Ga68. The usefulness of metal ions for both ln
vitro and in vivo diagnostic procedures is disclosed in
U.S. Patent 4,454,106.
The most desirable embodiment of this
diagnostic process uses Pb203 pb203 h
half-life as a gamma emitter. Pb203 has a unique
property in that it decays in a high percentage by
single photon emission. This gamma emission is
preferred and dominant over all other emissions. This
single photon emission makes Pb203 useful for single
photon emission computed tomography (SPECT) which is a
diagnostic tool. Thu~, when Pb203 is linked by use of
WO91/1~58 PCT/US91/01637
16
a chelate to a hapten, which specifically localizes in
a tumor, then that particular localization can be
dimensionally mapped for diagnostic purposes ln vivo by
SPECT. Alternatively, the emission can be used in
vitro in radioimmunoassay.
The present invention can be illustrated by
the use of the following non-limiting examples.
EXAMPLE 1
Svnthesis of ~-Nitrobenzyl 1 4.7 10
tetraazacyclododecane triacetic acids
The cyclen (compound 3 of Figure 1) was
alkylated at 45~C with three sequential additions of 1
equivalent of bromoacetic acid in the presence of 5 M
sodium hydroxide. The base was added via an
autoburette, and the pH of the reaction solution was
maintained at 8.5. The reaction was allowed to stir at
45-C overnight after the final addition. The following
day the solution was acidified to pH 2.0 with 3 N HCl.
The solution was loaded onto a 2.6 x 30 cm AG50wX8
200/400 mesh H+ form ion exchange column and washed
with water until the eluant was neutral. The crude
product was eluted form the column with one liter of 2
M NH40H. The solution was rotary evaporated to a
solid. The solid was taken up in water (Z5 mL total
with rinsing ) and loaded onto a 1.6 x 20 cm AGlwX8
200/400 mesh HOAc form ion exchange column. The
product was eluted with a two liter gradient of 0.0 to
0.3 M HOAc. The relevant fractions were combined,
concentrated to 25 mL, and then freeze dried to give a
fluffy white solid product.
WO9~ 8 PCT/US91/01637
2~9~3~
EXAMPLE 2
Labelinq of Antibod~ With Metal Ch~elate
The procedures and reagents described above
for the preferred embodiment of making the compounds
are used for this example.
The monoclonal antibody B72.3 binds
specifically to a glycoprotein on LS-174T cells. This
glycoprotein is also present in humans who have colon
cancer. This antibody is labeled with the ligand of
compound 5 of Figure 1 as follows:
One mL of B72.3 (10 mg/mL) was cooled in an
ice bath and combined with 20 ~L of 2.5 M sodium
acetate (pH 6). Sodium periodzte (NaIO4, 3.5-4.7 mg,
Aldrich) was added, giving a NaIO4 concentration of
16.22 mM. After the solid was dissolved, the clear
solution was left on ice in the refrigerator for 1 1/2
hrs. The solution was then passed through a 2 x 10 cm
Sephadex G-50 column, eluted with acetate-buffered
saline (pH 6). Ten fractions (1 mL each) were
collected, and the concentration of B72.3 in each
fraction was determined by W absorption at 220 nm.
The fractions containing oxidized B72.3 (6.7-9.0 mg in
2.2 mL) were collected and reacted with excess compound
5 at a molar ratio of 1:50-60. The mixture was mixed,
allowed to stand at room temperature for one hr, and
finally left in the refrigerator overnight. The next
day, sodium cyanoborohydride (Aldrich, 20-25 ~L of 1 M
solution) was added to the chelate-conjugated B72.3 to
reduce the Schiff base to the saturated amine. This
mixture was allowed to stand at room temperature for 4-
5 hr.
WO9~/l~58 PCT/US9ltO1637
2~792a~
Before labeling with metal, the protein is
dialyzed against a solution comprising 0.02 M N-
morpholinoethanesulfonic acid and 0.01 M NaCl at pH
5.9.
The protein in solution is labeled with Y90 by
reaction with an acetate solution of the isotope
followed by passage through a TSK 3000 size exclusion
column. This is a high pressure liquid chromatography
technique. The compound is mixed with an excipient and
lo is used in a model system in athvmic mice which have
been implanted with LS-174T cells to develop a tumor in
the flank of the animal. The antibody localizes
specifically to these tumor cells to deliver its
radiation.
EXAMPLE 3
In Vivo Use of the Labeled Protein Con~uaate
The protein conjugate in solution in labeled
with indium or gadolinium, and the chelate conjugate is
injected or introduced into body fluids of a mammal.
The antibody then localizes delivering the indium or
gadolinium to the tumor site, and conventional gamma
camera or magnetic resonance imaging techniques are
employed to respectively visualize the malignancy.
