Method for preparing radionuclide-labeled chelating agent-ligand complexes

Radionuclide-labeled chelating agent-ligand complexes that are useful in medical diagnosis or therapy are prepared by reacting a radionuclide, such as .sup.90 Y or .sup.111 In, with a polyfunctional chelating agent to form a radionuclide chelate that is electrically neutral; purifying the chelate by anion exchange chromatography; and reacting the purified chelate with a targeting molecule, such as a monoclonal antibody, to form the complex.

DESCRIPTION 
1. Technical Field 
This invention relates to a method for preparing radionuclide-containing 
compounds that are useful for medical diagnosis and therapy. 
2. Background Art 
Macrocyclic bifunctional chelating agents have been developed to tag 
monoclonal antibodies (mAbs) with radiometals for in vivo diagnosis and 
therapy Moi et al., 1985, Anal. Biochem., 148:249-253; Moi et al., 1988, 
J. Am. Chem. Soc., 110:6266-6267; Cox et al., 1990, J. Chem. Soc. Perkins 
Trans. 1, 2567-2576; Parker, 1990, Chem. Soc. Rev., 19:271-291; Meares et 
al., 1990, British J. Cancer, Suppl., 10:21-26; Gansow, 1991, Nucl. Med. 
Biol., 18:369-381; Li et al., 1993, Bioconjugate Chem., 4:275-283!. In 
particular, mAbs labeled with DOTA 
(1,4,7,10-tetraazacyclododecane-N,N',N",N'"-tetraacetic acid) derivatives 
incorporating yttrium-90 (.sup.90 Y) and indium-111 (.sup.111 In) have 
shown excellent kinetic stability under physiological conditions Moi et 
al., 1988; Meares et al., 1990; Li et al., 1993; Deshpande et al., 1990, 
J. Nucl. Med., 31:473-479!. However, the slow formation of yttrium-DOTA 
complexes Kapryzyk et al., 1982, Inorg. Chem., 21:3349-3352; Kodama et 
al., 1991, Inorg. Chem., 30:1270-1273; Wang et al., 1992, Inorg. Chem., 
31:1095-1099! presents a technical problem that can lead to low 
radiolabeling yields unless conditions are carefully controlled. 
These chelating agent-mAb-radionuclide conjugates have been synthesized 
using two methods. In one, the chelating agent is first conjugated to the 
antibody and then the resulting conjugate is labeled with radionuclide. In 
the other, called "prelabeling", the chelating agent is first labeled with 
the radionuclide, the labeled chelating agent is purified, and the 
purified labeled chelating agent is conjugated to the antibody. 
Prelabeling has several potential advantages over the other method. In the 
labeling step, metal chelate formation is easier to control because there 
is no competition from metal binding sites on the mAb and there is no 
danger of denaturing the antibody during labeling. The removal of 
unreacted chelating agent in the purification step avoids the production 
of multiply labeled immunoconjugates with unfavorable biological 
properties. Finally, prelabeling minimizes chemical manipulation of the 
antibody and reduces loss of antibody activity. 
Prelabeling has been used to label mAbs with .sup.99 Tc Fritzberg et al., 
1987, Proc. Natl. Acad. Sci. U.S.A., 85:4025-4029; Franz et al., 1987, 
Nucl. Med. Biol., 14:569-572; Linder et al., 1991, Bioconjugate Chem., 
2:160-170!, .sup.67 Cu Moi et al., 1985, supra! and .sup.177 Lu Schlom 
et al., 1991, Cancer Res., 51:2889-2896!. It has not heretofore been used 
to label mAbs with .sup.90 Y or .sup.111 In. 
In these prior instances of prelabeling, the metal chelate has either not 
been purified prior to conjugation Moi et al., 1985, supra! or has been 
purified by HPLC Fritzberg et al., 1987; Schlom et al., 1991, supra!. It 
is noted that Moi et al. use anion exchange chromatography to characterize 
their chelate (as a divalent anion) but not to purify it. The use of HPLC 
is not desirable because it employs mixed aqueous/organic solvents for 
elutions. 
DISCLOSURE OF THE INVENTION 
The present invention applies the prelabeling process to .sup.90 Y and 
.sup.111 indium labeling and provides a prelabeling process that employs 
anion exchange chromatography to purify the radionuclide chelate. 
Accordingly, one aspect of the invention is a method for preparing a 
yttrium- or indium-labeled chelating agent-ligand complex comprising: 
(a) reacting a chelating agent that has a trivalent chelating group and at 
least one pendant linker group that is capable of covalently binding to a 
ligand, with yttrium-90 or indium-111 to form an electrically neutral 
yttrium-90 or indium-111 chelate; 
(b) purifying the chelate from the reaction mixture of step (a); and 
(c) reacting the purified chelate of step (b) with the ligand to form said 
complex. 
Another aspect of the invention is a method for preparing a 
radionuclide-labeled chelating agent-ligand complex comprising: 
(a) reacting a chelating agent that has a chelating group and at least one 
pendant linker group that is capable of covalently binding a ligand, with 
a radionuclide to form a radionuclide chelate; 
(b) purifying the radionuclide chelate from the reaction mixture of step 
(a) by anion exchange chromatography; and 
(c) reacting the purified radionuclide chelate of step (b) with the ligand 
to form said complex.

DETAILED DESCRIPTION OF THE INVENTION 
A. The Novel Prelabeling Method 
Prelabeling involves three steps: (1) formation of a radiolabeled chelate 
(in the absence of ligand(s)), (2) purification of the radiolabeled 
chelate, and (3) conjugation of the purified radiolabeled chelate with 
ligand(s) to form a radiolabeled chelating agent-ligand complex. 
The medically useful radiometals which are important for practical 
applications have short half-lives Wessels et al., 1984, Med. Phys., 
11:638-645! and high efficiencies of both labeling and conjugation. The 
prelabeling approach permits use of a large excess of chelating agent to 
achieve a high chelation yield quickly in step (1), but requires a rapid 
purification method to remove unlabeled reagent in step (2). The present 
invention provides an easy and efficient method for prelabeling a 
chelating agent (for example, a peptide-linked DOTA derivative) with a 
radiometal (for example, .sup.90 Y or .sup.111 In) and subsequently 
conjugating it to a targeting molecule (for example, a mAb). 
In the conventional labeling method, the number of radionuclide chelating 
moieties attached to each targeting molecule is usually &gt;1 in order to 
provide enough chelating groups for a good radiolabeling yield. However, 
the chelating groups that actually chelate radionuclides comprise less 
than 5% of the total attached chelating groups on the targeting molecule. 
The excess chelating groups may affect the biological properties of the 
targeting molecule, e.g. by inducing an immune response Kosmas et al., 
1992, supra!, and impure metal solutions may require large amounts of the 
targeting molecule. 
With prelabeling, a far smaller number of chelates are attached to the 
targeting moiety, but practically all are radiolabeled; thus the number of 
modified targeting molecules is significantly reduced, and the number of 
multiply-modified targeting molecules is essentially zero. For example, 
when the targeting molecule is a mAb, the radiolabeled mAbs are fully 
immunoreactive and are expected to have more favorable biological 
properties, including reduced immunogenicity. 
B. Formation of the Radiolabeled Chelate 
The first step of prelabeling involves the formation of a 
radiolabeled-chelating agent (also referred to as a radiolabeled chelate) 
from a chelating agent and an appropriate radionuclide. 
Synthetic methods for the preparation of chelating agents useful in the 
practice of the invention are known in art see, for example Li et al., 
1993, supra!. 
Methods for the preparation of radiolabeled chelates by reaction of a 
radionuclide with a chelating agent are known in the art see, for 
example, Moi et al., 1985, supra!. Typically, the chelating agent is 
dissolved in a buffered aqueous medium and the purified radionuclide 
added. The pH may be selected to optimize conditions for chelate 
formation. For example, when chelation is achieved by acetate groups 
binding to the metal ion (as is the case for various acetic acid 
compounds), the pH may be adjusted (using, for example, aqueous 
tetramethylammonium acetate, to obtain of pH of about 3 to about 6, more 
preferably about 5) to provide a preponderance of ionized carboxylate 
(--COO.sup.-) groups, and thereby yield a chelating species which is 
anionic. Furthermore, the reaction mixture temperature may be adjusted, 
for example to 37.degree. C. for 30 min, to accelerate the reaction 
(chelation). After a period of time or upon completion of reaction, an 
excess of an appropriate quenching agent, such as DTPA may be added. The 
quenching agent acts to form anionic quenching chelates with any 
radionuclide not yet chelated by the chelating agent. The resulting 
reaction mixture may then be purified by the second step of prelabeling. 
The term "radionuclide", as used herein, relates to medically useful 
radionuclides, including, for example, positively charged ions of 
radiometals such as Y, In, Cu, Lu, Tc, Re, Co, Fe and the like, such as 
.sup.90 Y, .sup.111 In, .sup.67 Cu, .sup.77 Lu, .sup.99 Tc and the like, 
preferably trivalent cations, such as .sup.90 Y and .sup.111 In. 
The term "chelating agent", as used herein, relates to polyfunctional 
compounds have a chelating group and at least one pendant linker group, 
wherein the chelating group is capable of chelating with a radionuclide, 
and the pendant linker group(s) is capable of covalently binding to one or 
more targeting molecules which may be the same or different. Chelating 
agents may be represented by the formula A(L).sub.n wherein A represents 
the chelating moiety, L represents a pendant linking group, and n is an 
integer from 1 to 3, preferably 1. The pendant linker group includes one 
or more functional group(s) which are capable of covalently binding to 
targeting molecule(s). 
Chelating groups capable of chelating radionuclides include macrocycles, 
linear, or branched moieties. Examples of macrocyclic chelating moieties 
include polyaza- and polyoxamacrocycles. Examples of polyazamacrocyclic 
moieties include those derived from compounds such at 
1,4,7,10-tetraazacyclododecane-N,N',N",N'"-tetraacetic acid (herein 
abbreviated as DOTA); 
1,4,7,10-tetraazacyclotridecane-N,N',N",N'"-tetraacetic acid (herein 
abbreviated as TRITA); 
1,4,8,11-tetraazacyclotetradecane-N,N',N",N'"-tetraacetic acid (herein 
abbreviated as TETA); and 
1,5,9,13-tetraazacyclohexadecane-N,N',N",N'"-tetraacetic acid (abbreviated 
herein abbreviated as HETA). Examples of linear or branched chelating 
moieties include those derived from compounds such as 
ethylenediaminetetraacetic acid (herein abbreviated as EDTA) and 
diethylenetriaminepentaacetic acid (herein abbreviated as DTPA). 
Chelating moieties having carboxylic acid groups, such as DOTA, TRITA, 
HETA, HEXA, EDTA, and DTPA, may be derivatized to convert one or more 
carboxylic acid groups to amide groups. 
The term "pendant linker group", as used herein, relates to moieties which 
are attached to the chelating group, and which have at least one 
functional group which is capable of covalently binding to targeting 
molecules. Where pendant linkers or chelating agents have a plurality of 
such functional groups, they may be the same or different. When the 
chelating moiety is macrocyclic, the pendant moiety may be attached to any 
annular atom. For example, when the chelating moiety is a 
polyazamacrocycle, the pendant group may be attached to an annular carbon 
atom or an annular nitrogen atom. When the pendant group is attached to an 
annular nitrogen atom, the compound may be referred to as an N-substituted 
polyazamacrocycle. 
The term "functional groups capable of covalently binding to targeting 
molecules", as used herein, includes those functional groups which can be 
activated by known methods, so as to be capable of covalently binding to 
targeting molecule(s); for example, the formation of active esters 
(--C(.dbd.O)OR, wherein R is, for example, succinimidyl) from carboxylic 
acids, the formation of acid halides (--C(.dbd.O)X, wherein X is typically 
Cl or Br) from carboxylic acids. 
The functional group(s) present on the pendant linker group which are 
capable of covalently binding to targeting molecules may be chosen 
according to the targeting molecule(s) to which the chelating agent will 
ultimately be bound. Reactive pairs of functional groups permit 
conjugation of the chelating agent with the targeting molecule, via the 
linker group, wherein one member of the pair is present on the chelating 
agent and the other member of the pair is present on the targeting 
molecule. For example, when the targeting molecule is a protein possessing 
a free amino (--NH.sub.2) group, a functional group such as isothiocyanate 
(--NCS) present on the chelating agent permits reaction to form a joining 
linkage (in this case, a thiourea linkage), thereby forming a chelating 
agent-targeting molecule complex. Other examples of appropriate reactive 
pairs of functional groups include, for example, --NH.sub.2 with 
--C(.dbd.O)OR (active ester) or with --C(.dbd.O)OC(.dbd.O)R (anhydride) or 
with --C(.dbd.O)X (acid halide) to yield an amide linkage; --NH.sub.2 with 
--NCO (isocyanate) to yield a urea linkage. Other reactive pairs involving 
--NH.sub.2 include --NH.sub.2 and --S(.dbd.O).sub.2 X (sulfonyl halide); 
--NH.sub.2 and --C(.dbd.NR)OR (imidate ester); and --NH.sub.2 and 
--OC(.dbd.O)X (haloformate). Examples of reactive pairs of functional 
groups include --SH and --C(.dbd.O)CH.sub.2 X (haloacetyl) to yield a 
--SCH.sub.2 C(.dbd.O)-- linkage; --SH and -alkyl-X (alkyl halide) or --SH 
and --S(.dbd.O)O-alkyl (alkyl sulfonate) to yield a thioether; and --SH 
and --SH (sulfhydryl) to yield a --SS-- (disulfide) linkage. 
Li et al. Li et al., 1993, supra! have shown that the introduction of a 
cleavable linker between the chelate and the mAb results in a reduction of 
accumulated radioactivity in the liver Li et al., 1993, supra!. 
Preferably, radionuclide-labeled chelating agent-ligand complexes will be 
cleavable in vivo. This may be achieved by introducing a cleavable linkage 
within the pendant group, wherein the cleavable linkage is cleaved in 
vivo, for example, by enzymatic action within the liver. Examples of such 
cleavable linkages include peptide, disulfide, and ester linkages. 
Examples of pendant linker groups include peptide-based linkers, such as 
polypeptide groups which have been derivatized to possess at least one 
functional group capable of covalently binding to targeting molecule(s). 
The number of peptide linkages present in the pendant linker group may be 
varied to optimize radionuclide chelation, conjugation with targeting 
molecule(s), in vivo cleavability, or other factors. Examples of suitable 
pendant linker groups include --CH.sub.2 --C(.dbd.O) (AA).sub.m (AA-FG), 
herein denoted as substituted acetyls, wherein the --CH.sub.2 
--C(.dbd.O)-- fragment may be derived from an acetate moiety, AA 
represents an amino acid diradical, more preferably the glycine diradical 
--NH--CH.sub.2 --C(.dbd.O)--, and m is an integer, preferably between 1 
and 10, more preferably between 3 and 7, most preferably 3. AA-FG 
represents an amino acid N-radical (that is, the free bond is situated on 
the amino group of the amino acid) which has been derivatized to possess a 
functional group (FG) capable of covalently binding to targeting 
molecule(s). Preferably, the carboxylic acid group of the amino acid of 
AA-FG has been derivatized, for example, to form an amide. Examples of 
AA-FG radicals include p-isothioscyanato-phenylalanine-N-yl amide 
(--NHCHC(.dbd.O)NH.sub.2 !CH.sub.2 --(p-NCS--C.sub.6 H.sub.4)! denoted 
herein as p-NCS-Phe-amide, or p-NCS-L-Phe-amide). Examples of pendant 
groups include --CH.sub.2 --C(.dbd.O)(Gly).sub.m (p-NCS-Phe-amide), 
denoted herein as Gly.sub.m (p-NCS-Phe-amide)acetyl. Further examples of 
pendant linker groups include disulfides, such as alkyl disulfides 
including --CH.sub.2 --C(.dbd.O)--(CH.sub.2).sub.p SS(CH.sub.2).sub.q NS 
and the like, and esters, such as --CH.sub.2 --C(.dbd.O)--(CH.sub.2).sub.p 
C(.dbd.O)O(CH.sub.2).sub.q NSC and the like, wherein p and q are integers 
from about 1 to about 8. 
Examples of chelating agents include: 
1,4,7,10-tetraazacyclododecane-N-(Gly.sub.3 
(p-NCS-Phe-amide)acetyl)-N',N",N'"-triacetic acid; 
1,4,7,10-tetraazacyclotridecane-N-(Gly.sub.3 
(p-NCS-Phe-amide)acetyl)-N',N",N'"-triacetic acid; 
1,4,8,11-tetraazacyclotetradecane-N-(Gly.sub.3 
(p-NCS-Phe-amide)acetyl)-N',N",N'"-triacetic acid; 
1,5,9,13-tetraazacyclohexadecane-N-(Gly.sub.3 
(p-NCS-Phe-amide)acetyl)-N',N",N'"-triacetic acid; 
ethylenediamine-N-(Gly.sub.3 (p-NCS-Phe-amide)acetyl)triacetic acid; 
diethylenetriamine-N-(Gly.sub.3 
(p-NCS-Phe-amide)acetyl)-N'-acetamide-N",N'",N""-triacetic acid; 
1,4,7,10-tetraazacyclododecane-N-(Gly.sub.2 
(p-NCS-Phe-amide)acetyl)-N',N",N'"-triacetic acid; 
1,4,7,10-tetraazacyclododecane-N-(Gly.sub.4 
(p-NCS-Phe-amide)acetyl)-N',N",N'"-triacetic acid; 
1,4,7,10-tetraazacyclododecane-N-(Gly.sub.5 
(p-NCS-Phe-amide)acetyl)-N',N",N'"-triacetic acid; 
1,4,7,10-tetraazacyclododecane-N-(Gly.sub.6 
(p-NCS-Phe-amide)acetyl)-N',N",N'"-triacetic acid; 
1,4,7,10-tetraazacyclododecane-N,N'-di(Gly.sub.3 
(p-NCS-Phe-amide)acetyl)-N",N'"-diacetic acid; 
1,4,7,10-tetraazacyclotridecane-N,N'-di(Gly.sub.3 
(p-NCS-Phe-amide)acetyl)-N",N'"-diacetic acid; 
1,4,8,11-tetraazacyclotetradecane-N,N'-di(Gly.sub.3 
(p-NCS-Phe-amide)acetyl)-N",N'"-diacetic acid; 
1,5,9,13-tetraazacyclohexadecane-N,N'-di(Gly.sub.3 
(p-NCS-Phe-amide)acetyl)-N",N'"-diacetic acid; 
ethylenediamine-N,N'-di(Gly.sub.3 (p-NCS-Phe-amide)acetyl)-N",N'"-diacetic 
acid; 
diethylenetriamine-N,N'-di(Gly.sub.3 
(p-NCS-Phe-amide)acetyl)-N"-acetamide-N'",N""-diacetic acid; 
1,4,7,10-tetraazacyclododecane-N-(Gly.sub.3 
(p-NCS-Phe-amide)acetyl)-N'-acetamide-N",N'"-diacetic acid; 
1,4,7,10-tetraazacyclotridecane-N-(Gly.sub.3 
(p-NCS-Phe-amide)acetyl)-N'-acetamide-,N",N'"-diacetic acid; 
1,4,8,11-tetraazacyclotetradecane-N-(Gly.sub.3 
(p-NCS-Phe-amide)acetyl)-N'-acetamide-N",N'"-diacetic acid; 
1,5,9,13-tetraazacyclohexadecane-N-(Gly.sub.3 
(p-NCS-Phe-amide)acetyl)-N'-acetamide-N",N'"-diacetic acid; 
ethylenediamine-N-(Gly.sub.3 
(p-NCS-Phe-amide)acetyl)-N'-acetamide-N",N'"-diacetic acid; 
diethylenetriamine-N-(Gly.sub.3 
(p-NCS-Phe-amide)acetyl)-N',N"-diacetamide-N'",N""-diacetic acid; 
1,4,7,10-tetraazacyclododecane-N-(Gly.sub.3 
(p-SH-Phe-amide)acetyl)-N',N",N'"-triacetic acid; 
1,4,7,10-tetraazacyclododecane-N-(Gly.sub.3 (p-succinimidyl 
ester-Phe-amide)acetyl)-N',N",N'"-triacetic acid; 
1,4,7,10-tetraazacyclododecane-N-(Gly.sub.3 
(p-chloroformyl-Phe-amide)acetyl)-N',N",N'"-triacetic acid; and 
1,4,7,10-tetraazacyclododecane-N-(Gly.sub.3 
(p-chloroacetyl-Phe-amide)acetyl)-N',N",N'"-triacetic acid. 
Examples of radiolabeled chelates include: 
.sup.90 Y.sup.III -1,4,7,10-tetraazacyclododecane-N-(Gly.sub.3 
(p-NCS-Phe-amide)acetyl)-N',N",N'"-triacetate; 
.sup.90 Y.sup.III -1,4,7,10-tetraazacyclotridecane-N-(Gly.sub.3 
(p-NCS-Phe-amide)acetyl)-N',N",N'"-triacetate; 
.sup.90 Y.sup.III -1,4,8,11-tetraazacyclotetradecane-N-(Gly.sub.3 
(p-NCS-Phe-amide)acetyl)-N', N",N'"-triacetate; 
.sup.90 Y.sup.III -1,5,9,13-tetraazacyclohexadecane-N-(Gly.sub.3 
(p-NCS-Phe-amide)acetyl)-N', N",N'"-triacetate; 
.sup.90 Y.sup.III -ethylenediamine-N-(Gly.sub.3 
(p-NCS-Phe-amide)acetyl)triacetate; 
.sup.90 Y.sup.III -diethylenetriamine-N-(Gly.sub.3 
(p-NCS-Phe-amide)acetyl)-N'-acetamide-N",N'",N""-triacetate; 
.sup.111 In.sup.III -1,4,7,10-tetraazacyclododecane-N-(Gly.sub.3 
(p-NCS-Phe-amide)acetyl)-N',N",N'"-triacetate; 
.sup.111 In.sup.III -1,4,7,10-tetraazacyclotridecane-N-(Gly.sub.3 
(p-NCS-Phe-amide)acetyl)-N',N",N'"-triacetate; 
.sup.111 In.sup.III -1,4,8,11-tetraazacyclotetradecane-N-(Gly.sub.3 
(p-NCS-Phe-amide)acetyl)-N',N",N'"-triacetate; 
.sup.111 In.sup.III -1,5,9,13-tetraazacyclohexadecane-N-(Gly.sub.3 
(p-NCS-Phe-amide)acetyl)-N',N",N'"-triacetate; 
.sup.111 In.sup.III -ethylenediamine-N-(Gly.sub.3 
(p-NCS-Phe-amide)acetyl)triacetate; 
.sup.111 In.sup.III -diethylenetriamine-N-(Gly.sub.3 
(p-NCS-Phe-amide)acetyl)-N'-acetamide-N",N'",N""-triacetate; 
.sup.67 Cu.sup.II -1,4,7,10-tetraazacyclododecane-N,N'-di(Gly.sub.3 
(p-NCS-Phe-amide)acetyl)-N",N'"-diacetate; 
.sup.67 Cu.sup.II -1,4,7,10-tetraazacyclotridecane-N,N'-di(Gly.sub.3 
(p-NCS-Phe-amide)acetyl)-N",N'"-diacetate; 
.sup.67 Cu.sup.II -1,4,8,11-tetraazacyclotetradecane-N,N'-di(Gly.sub.3 
(p-NCS-Phe-amide)acetyl)-N",N'"-diacetate; 
.sup.67 Cu.sup.II -1,5,9,13-tetraazacyclohexadecane-N,N'-di(Gly.sub.3 
(p-NCS-Phe-amide)acetyl)-N",N'"-diacetate; 
.sup.67 Cu.sup.II -ethylenediamine-N,N'-di(Gly.sub.3 
(p-NCS-Phe-amide)acetyl)-N",N'"-diacetate; 
.sup.67 Cu.sup.II -diethylenetriamine-N,N'-di(Gly.sub.3 
(p-NCS-Phe-amide)acetyl)-N"-acetamide-N'",N""-diacetate; 
.sup.67 Cu.sup.II -1,4,7,10-tetraazacyclododecane-N-(Gly.sub.3 
(p-NCS-Phe-amide)acetyl)-N'-acetamide-N",N'"-diacetate; 
.sup.67 Cu.sup.II -1,4,7,10-tetraazacyclotridecane-N-(Gly.sub.3 
(p-NCS-Phe-amide)acetyl)-N'-acetamide-,N",N'"-diacetate; 
.sup.67 Cu.sup.II -1,4,8,11-tetraazacyclotetradecane-N-(Gly.sub.3 
(p-NCS-Phe-amide)acetyl)-N'-acetamide-N",N'"-diacetate; 
.sup.67 Cu.sup.II -1,5,9,13-tetraazacyclohexadecane-N-(Gly.sub.3 
(p-NCS-Phe-amide)acetyl)-N'-acetamide-N",N'"-diacetate; 
.sup.67 Cu.sup.II -ethylenediamine-N-(Gly.sub.3 
(p-NCS-Phe-amide)acetyl)-N'-acetamide-N",N'"-diacetate; and 
.sup.67 Cu.sup.II -diethylenetriamine-N-(Gly.sub.3 
(p-NCS-Phe-amide)acetyl)-N',N"-diacetamide-N'",N""-diacetate. 
C. Purification of the Radiolabeled Chelate 
The second step of prelabeling involves purification of the radiolabeled 
chelate. The aqueous chelating agent-radionuclide reaction mixture 
obtained in the first step of prelabeling may be purified by preparing an 
appropriate anion exchange medium, eluting the aqueous mixture through the 
medium, and collecting the eluent. Methods for preparing and using anion 
exchange media are well established and known to those skilled in the art. 
If necessary or desired, the eluent may be further concentrated by known 
methods. 
The use of the well established technique of anion exchange chromatography 
to purify the radiolabeled chelate offers a number of practical advantages 
over other known methods of purification. 
For example, the aqueous reaction mixture obtained in the first step of 
prelabeling may be purified simply and quickly by anion exchange 
chromatography to provide the purified compound in aqueous solution and in 
the absence of organic solvents. Consequently, there is no need to remove 
organic solvents from the purified material before proceeding with 
formation of the radiolabeled-chelating agent-ligand complex, as required 
by the existing prelabeling methods see, for example, Linder et al., 
1991; Schlom et al., 1991, supra!. The anion exchange chromatography 
purification step of the present invention saves time and thereby reduces 
the loss of radioactive potency and minimizes autoradiolysis. 
Purification of the chelate by anion exchange chromatography can be 
significantly improved by selecting chelating agents and radionuclides 
with equal and opposite electrical charges. The resulting radiolabeled 
chelate is electrically neutral. The other important species in the 
chelation reaction mixture, such as excess chelating agent, complexes 
containing differently charged metal ions, and quenching agent complexes, 
are negatively charged. Thus, the electrically neutral radiolabeled 
chelate of interest can be filtered quickly through an appropriately 
designed anion-exchange column in H.sub.2 O to separate them from anionic 
species. Purification can be further improved by selecting chelating 
agent/radionuclide charges of -3/+3, so that chelates formed from 
adventitious cations, such as Ca.sup.+2 and Mg.sup.+2 are anionic. 
Any appropriately prepared anion exchange material may be used to effect 
purification of the radiolabeled chelate. Commercially available (Sigma 
Chemical Company) anion exchange media include, for example, dextran-based 
anion exchange media such as DEAE Sephadex and QAE Sephadex, agarose-based 
anion exchange media such as DEAE Sepharose and Q Sepharose, 
cellulose-based anion exchange media, such as DEAE Sephacel, DEAE 
Cellulose, ECTEOLA Cellulose, PEI Cellulose, QAE Cellulose, and 
polystyrene-based anion exchange media, such as Amberlite and Dowex. 
Criteria for choosing and methods of preparation of anion exchange media 
are well established and known to those of skill in the art. At present, 
the preferred anion exchange medium is DEAE Cellulose prepared in acetate 
form. 
D. Formation of the Complex 
The third step of prelabeling, conjugation, involves the formation of a 
radiolabeled-chelating agent-ligand complex (denoted herein variously as 
the chelate-ligand complex or the complex). 
The term "ligand", as used herein, relates to compounds which may perform 
the role of targeting molecules, including, for example, targeting 
biomolecules such as antibodies, serum proteins, cell surface receptors, 
and tumor-specific targeting agents, such as bleomycin. The term 
"antibody" as used herein is generic to polyclonal antibodies, monoclonal 
antibodies, chimeric antibodies, antibody fragments (particularly antibody 
binding fragments), recombinant single chain antibody fragments, and the 
like. Methods for the preparation of appropriate ligands, such as mAbs, 
are well established and known to those of skill in the art See, for 
example, Fell et al., 1992, supra!. 
Reaction conditions useful for the formation of the complex from the 
radiolabeled chelate and ligand, such as pH, temperature, salt 
concentration, and the like, will reflect the reactive pair of functional 
groups involved. In particular, reaction conditions useful for the 
formation of conjugates via functional groups present on proteins are well 
established and known to those of skill in the art. Typically, aqueous 
solutions or suspensions of the radiolabeled chelate and the ligand are 
mixed. The pH may be adjusted to optimize conditions for conjugation. For 
example, for reaction of the amino (--NH.sub.2) group of a protein (for 
example, a mAb) with an isothiocyanato (--NCS) group, the pH may be 
adjusted to about 8 to 11, more preferably about 9.5, using, for example, 
a buffer such as aqueous triethylamine. Furthermore, the temperature of 
the reaction mixture may be adjusted, for example, to 37.degree. C. for 1 
hr, to accelerate conjugation. The reaction (incubation) time may be 
varied to optimize conjugation. Longer reaction times would lead to higher 
conjugation yields; however, for radioactivity levels appropriate for 
clinical use (.apprxeq.100 millicuries, mCi), radiolysis will become 
important at longer times. 
In the conjugation step, a high concentration of ligand (for example, mAb) 
is desired, so that each reactive functional group present on the 
radiolabeled-chelate (for example, isothiocyanate groups) will frequently 
encounter reactive functional groups on the ligand (for example, amino 
groups) with which to react. It may be desirable to concentrate the 
radiolabeled chelate prior to the conjugation step to avoid dilution of 
the conjugation mixture, particularly when small amounts of radioactivity 
are used. Optimally, ligand concentrations as high as are practical are 
desired, for example, about 10 to 500 mg/mL (the reaction mixture 
concentration of the chimeric mAb 16 ligand of Example 3 was about 20 
mg/mL). 
After conjugation, the complex may be separated from the reaction mixture 
and purified using known methods, for example, using a centrifuged 
gel-filtration column Penefsky et al., 1979, Methods Enzymol., 56, Part 
G:527-530; Meares et al., 1984, Anal. Biochem., 142:68-78!. 
E. Use 
One application of the radionuclide-labeled chelating agent-ligand 
complexes of the invention is for use in radioimaging. A variety of metal 
chelates, when conjugated with serum proteins, target-specific antibodies, 
or bleomycin can be localized in tumor or other target tissue to provide 
useful radioimaging images used in localizing tumors Meares et al., 1984; 
Goodwin et al., 1979, Radiopharmaceuticals II, Proceedings of the Second 
International Symposium on Radiopharmaceuticals (V. J. Sodd, ed), New 
York, pp.275-284; De Reimer et al., J. Lab. Comps. and Radiopharm., 
18(10):1517; Meares et al., 1976, Proc. Natl. Acad. Sci. U.S.A., 
73(11):3803; Leung et al., 1978, Int. J. Appl. Rad. Isot., 29:687; Goodwin 
et al., 1981, J. Nucl. Med., 22(9):787!. For example, a composition 
comprising the radionuclide-labelled chelating agent-ligand complex and a 
pharmaceutically acceptable carrier is injected in a patient and allowed 
to localize, for example, in a tumor region(s). These regions are imaged 
using radioimaging equipment such as .gamma. photon emission tomograph or 
a position emission computed tomograph. Various stratagems may be used to 
enhance the image contrast which is achievable. For example, with a 
complex formed with a serum protein, the image contrast can be improved by 
administering an anti-complex antibody following tumor uptake of the 
complex, to increase the rate of clearance of the complex from the 
bloodstream Goodwin et al., 1981, supra!. 
The radionuclide-labeled chelating agent-ligand complexes of the invention 
are also suitable for use as therapeutic agents based, for example, on the 
radiotherapeutic action of the radionuclide when localized in tumor 
tissue. Again, a composition comprising the radionuclide-labelled 
chelating agent-ligand complex and a pharmaceutically acceptable carrier 
is injected in a patient and allowed to localize, for example, in a tumor 
region(s). 
F. Examples 
The following examples further illustrate the invention. These examples are 
not intended to limit the invention in any manner. 
Example 1 
Preparation of the .sup.90 Y-Labeled Chelating Agent (2) 
The prelabeling procedure is shown in FIG. 1. (Bold letters in parentheses 
refer to the compounds shown in the figures). The bifunctional chelating 
agent (1) (denoted herein variously as DOTA-Gly.sub.3 
-L-(p-isothiocyanato)-Phe-amide and 
1,4,7,10-tetraazacyclododecane-N-(Gly.sub.3 
(L-p-NCS-Phe-amide)acetyl)-N',N",N'"-triacetic acid) was prepared by the 
method described by Li et al., 1993, Bioconjugate Chem., 4:275-283. 
Carrier-free .sup.90 Y (DuPont NEN) in 0.05 M HCl was dried in a heating 
block under N.sub.2 (g), and 100 .mu.L of 20 mM (1) in 0.2 M 
(CH.sub.3).sub.4 N.sup.+ acetate, pH 5.0, was added. The mixture was 
incubated at 37.degree. C. for 30 min, followed by the addition of 25 
.mu.L of 50 Mm DTPA in 0.1 M (CH.sub.3).sub.4 N.sup.+ acetate, pH 6.0, 
for 15 min at room temperature (quenching; to complex any remaining free 
yttrium). 
An anion-exchange column was prepared by filling a disposable 1 mL 
tuberculin syringe with 500 .mu.L of DEAE (diethylaminoethyl) Cellulose 
anion-exchange resin (1 milliequivalent per dry gram; Sigma Chemical 
Company) and pre-spun for 3 min at .apprxeq.2000 g. The resin was 
converted to acetate form prior to use. 
The solution was loaded onto the anion-exchange column, and the column was 
spun for 2 min at .apprxeq.2000 g. followed by elution with four 125 .mu.L 
aliquots of H.sub.2 O by centrifugation at .apprxeq.2000 g for 2 min each. 
Most of the radioactive compound (2) (denoted herein variously as .sup.90 
Y.sup.III -DOTA-Gly.sub.3 -L-(p-isothiocyanato)-Phe-amide and .sup.90 
Y.sup.III -1,4,7,10-tetraazacyclododecane-N-(Gly.sub.3 
(L-p-NCS-Phe-amide)acetyl)-N',N",N'"-triacetate) was recovered in the 
first four fractions (see Table 1). One-step elution with 0.5 mL of 
H.sub.2 O was performed for comparison, but it gave 18% lower recovery 
than stepwise elution with 0.5 mL of H.sub.2 O was performed for 
comparison, but it gave 18% lower recovery then stepwise elution. 
All the eluted fractions were collected and concentrated to .apprxeq.15 
.mu.L with a speed-vac concentrator (Savant Instruments) without heating. 
It should be possible to avoid this step when larger amounts of 
radioactivity are used. 
In the chelation step, the yield after anion-exchange was typically &gt;70% of 
the starting radioactivity. Particularly for .sup.90 Y solutions, the 
levels of metal impurities appear to vary with each batch of carrier-free 
radiometal. The identity of these impurities is difficult to determine, 
but most common metal contaminants are divalent ions. Pre-labeling deals 
with the impurity problem by using a large excess of chelating agent, and 
then removing the excess. This is preferable to using a large excess of 
chelate-tagged mAb conjugate, which cannot be fractionated later to remove 
unwanted contaminants. Obviously, pre-labeling does not eliminate 
trivalent metal complexes from the product. 
Example 2 
Preparation of the .sup.111 In-Labeled Chelating Agent (2) 
.sup.111 In-labeled chelate (2) was prepared by the general procedure of 
Example 1. 
Example 3 
Preparation of the Radionuclide-Labeled Chelating Agent-Targeting Molecule 
Complex (3) 
Concentrated solutions of the labeled chelating agent of Example 1 (and 
alternatively of Example 2) were mixed with 1 mg of chimeric mAb 16 (18 
.mu.L, 56 mg/mL; Oncogen/Bristol-Myers) Fell et al., 1992, J. Biol. 
Chem., 267:15552-15558! in 0.1 M (CH.sub.3).sub.4 N.sup.+ phosphate, pH 
9.0. The pH was adjusted to 9.5 using aqueous 2.0 M triethylamine. The 
mixture was incubated at 37.degree. C. for 1 hr and compound (3) was 
isolated using a centrifuged gel-filtration column Penefsky et al., 1979; 
Meares et al. 1984, supra!. Yields are listed in Table 1. 
TABLE 1 
______________________________________ 
Results for DOTA-Peptide Radiolabeling and Conjugation 
Starting Recovery for 
Recovery for 
Radioactivity 
Step 2.dagger. 
Step 3.dagger. 
Overall 
Radionuclide 
(volume) (radiolabeling) 
(conjugation) 
Recovery.dagger. 
______________________________________ 
.sup.90 Y 
2.1-3.9 mCi 
80% .+-. 5% 
40% .+-. 2% 
30% .+-. 4% 
(2-5 .mu.L) 
.sup.111 In 
4.9-6.2 mCi 
70% .+-. 9% 
73 .+-. 3% 
42% .+-. 4% 
(12-30 .mu.L) 
______________________________________ 
.dagger.Average recovered radioactivity .+-. standard deviation, for 
.gtoreq.3 runs. 
The radiochemical purity of both .sup.90 Y-and .sup.111 In-labeled 
immunoconjugates (3) was determined to be &gt;95% by gel filtration HPLC 
cellulose acetate electrophoresis, and silica gel TLC Meares et al., 
1984, supra!. A solid-phase radioimmunoassay DeNardo et al., 1986, Nucl. 
Med. Biol., 13:303-310! was performed using .sup.125 I-labeled chimeric L6 
as a standard. The immunoreactivity of .sup.90 Y-DOTA-Gly.sub.3 
-L-Phe-amide-thiourea-chimeric L6 was 107.+-.5% relative to .sup.125 
I-labeled antibody. 
Example 4 
Biodistribution of the Radionuclide-Labeled Chelating Agent-Targeting 
Molecule Complex 
To examine the properties of the conjugate in vivo, .sup.90 Y-labeled 
compound (3) was injected into HBT tumor-bearing nude mice Hellstrom et 
al., 1986, Cancer Res., 46:3917-3923! for organ distribution and tumor 
uptake studies. The results of these animal studies (summarized in FIG. 2) 
showed that the radioactivity level in the liver varied from 6.4.+-.1.5% 
I.D./g on the first day to 5.4.+-.1.5% I.D./g on the third day to 
4.6.+-.1.9% I.D./g on the fifth day. The units I.D./g indicate percent of 
injected dose per gram of tissue. The tumor uptake was 17.5.+-.8.0% I.D./g 
on day 1, 18.0.+-.8.0% I.D./g on day 3 and 13.8%.+-.5.2% I.D./g on day 5. 
The bone uptake was 2.1.+-.0.3% I.D./g, 2.0.+-.0.5% I.D./g, and 
1.8.+-.0.2% I.D./g on days 1, 3, and 5. The levels of radioactivity in 
liver and bone are satisfactorily low Deshpande et al., 1990, supra!, and 
the tumor uptake is good. 
All publications, patents, and patent applications cited in this 
specification are herein incorporated by reference as if each individual 
publication, patent, or patent application were specifically and 
individually indicated to be incorporated by reference.