Method for preparing .sup.213 Bi for therapeutic use

The present invention relates to the field of site directed therapy. More specifically it relates to site directed radio therapy. It provides a method for production of radioimmuno conjugates and an apparatus for radioimmuno therapy. The method, conjugates and apparatus can be practicalized without the need for radioactive shielding and/or airtight facilities. Without these restrictions the invention provides a simple and efficient means of therapy at the bed-side of the patient.

The present invention relates to the field of site directed therapy. 
Nowadays there are a number of methods of site directed therapy which have 
been suggested to eliminate unwanted cells or infectious organisms from 
the body of a mammalian subject. 
There are many fields of therapy in which said methods may be applied. 
The most important ones seem to be immune diseases (either auto immune 
diseases or acquired immune diseases), cancer and viral or microbial 
infections. 
BACKGROUND OF THE INVENTION 
Site directed therapy is a method whereby a cytotoxic compound is delivered 
to the immediate vicinity of the target cell or infectious organism. This 
is usually done by coupling a targeting moiety to the cytotoxic compound. 
This targeting moiety recognizes a structure in, on, or near the target. 
Known targeting moieties include, but are not limited to, antibodies, more 
specifically monoclonal antibodies and more preferably human monoclonal 
antibodies, nucleic acids, receptor directed ligands and the like. 
Cytotoxic compounds can be for instance drugs, such as adriamycin, toxins 
such as ricin A and radioisotopes. 
Radioisotopes cannot only be used for therapy, but they can also be used to 
identify the site or sites of the target (imaging). This invention 
provides methods of therapy and imaging using a conjugate of a targeting 
moiety and at least one radioisotope. 
Therapy with targeting moieties is widely known. Targeting can be 
accomplished by aiming the targeting moiety directly to the wanted site, 
but it may also be directed to another targeting moiety which is directed 
to the wanted site (so called pretargeting). Pretargeting offers an 
advantage over direct targeting when the specificity of the targeting 
moieties is not sufficient. By using a first localizing moiety followed by 
a second one coupled to a cytotoxic compound, the amount of cytotoxic 
compound delivered to non-target sites can be lowered significantly. 
Known targeting moieties, such as antibodies, often cannot be provided with 
a large amount of cytotoxic compounds without hampering their targeting 
specificity. 
Therefore it has often been suggested to use a carrier molecule, such as 
HSA or a nucleic acid, or a polymer, which can be loaded with a high 
number of cytotoxic compounds and coupled to a targeting moiety. 
All of the above-mentioned variations on the theme of site directed therapy 
and/or imaging can be used more advantageously with the present invention. 
A well established problem in the field of imaging and site directed 
radiotherapy is to find a suitable radioisotope. Apart from the amount of 
energy that is released upon their decay, which should be sufficient to be 
measurable outside the subject in the case of imaging and sufficiently 
lethal to the target in the case of therapy, there is also a problem in 
finding an isotope with a suitable half-life. 
An isotope with a long half life cannot be chosen because of the biological 
half life of the targeting moiety, which means that most of the isotopes 
will decay after disintegration of the conjugate. This decay after the 
disintegration of the conjugate will lead to cytotoxicity to other cells 
or tissues than the target. 
Furthermore, all conjugates which do not localize will be secreted from the 
body and present a radio active waste problem. 
It is also not practical to choose a radioisotope with too short a half 
life, because of packing and shipping delays and because the institution 
carrying out the therapy must be equipped to make the conjugate, transport 
it to the patient and administer it in a very short interval of time, 
otherwise most of the radioisotope will have decayed before entering the 
body, let alone localization at the target site. 
The isotopes used for imaging usually are gamma emitting isotopes, for 
therapy auger electron emitting .alpha.,.beta.-emitting, or 
.alpha.-emitting isotopes may be used. 
Most preferred for the present invention are .alpha.-emitting isotopes. 
The short-range cell-killing effect of .alpha.-particles is enormous: a 1 
mm diameter tumor, comprising maybe 600,000 cancer cells needs about 6 
.alpha.-particles of 6 MeV per cell to deliver a dose of 600 rad, causing 
a 99.9% cell-kill ratio, and that specifically because of the stochastic 
nature of the hit- and kill-mechanism. 
However, due to the same stochastic nature, a 10 times lower 
.alpha.-radiaton dose will enhance the cell-survival ratio with a factor 
500: more than 50% of the cells (or non-tumor cells in similar morphology 
for that matter) would survive a 60 rad .alpha.-radiation dose, equivalent 
to 0.6 .alpha.-particles per cell. 
This characteristic would make an effective .alpha.-radioimmunotherapy 
within reach, provided that a "quality factor" for the isotope-antibody 
conjugate of 10 or better can be obained. It is the purpose of the present 
invention to contribute towards this goal in a most essential manner. 
The quality factor is a ratio between localized antibody at the target 
site, divided by the antibody "sticking" to other tissue. 
The notion of using .alpha.-particles emanating radioisotopes as agents for 
the killing of tumor cells was already mentioned in the literature during 
the mid-fifties. Since then other potential candidate-isotopes were and 
are being proposed, of which a good summary is given by Fisher (1) and by 
Wilbur (2) which brings the list to (with their half-lifes between 
parentheses): .sup.223 Ra (11.4 d), .sup.225 Ac (10 d), .sup.224 Ra (3.6 
d), .sup.225 Fm (20 h), .sup.211 At (7,2 h), .sup.212 Bi (60 m), and 
.sup.213 Bi (47 m). 
Although important publications appear regularly in the literature 
regarding microdosimetry, antibody-isotope coupling techniques, 
pre-clinical in vitro and in vivo experiments, no clearly defined, larger 
scale clinical experiments are being done until now, for a variety of 
reasons: 
a. no human monoclonal antibodies with proven sufficient quality are 
available yet, 
b. no biological safety data are available for antibody coupling agent (the 
latter for the binding of the radioisotope) combinations, 
c. some isotopes may not become available for large scale application at 
acceptable costprices (.sup.225 Fm), 
d. isotopes may be too difficult and therefore too expensive to obtain 
because of the necessary procurement process (.sup.211 At from .sup.209 Bi 
by a (.alpha., 2n) reaction in a cyclotron and subsequent isolation plus 
purification), 
e. other isotopes do have a Rn-isotope as first daughter in their decay 
sequence, allowing redistribution of daughter nuclei before decay 
(.sup.224 Ra, .sup.223 Ra), and also necessitating gas-tight reaction 
conditions, 
f. some isotopes may have a relatively long living daughter isotope 
somewhere in their decay sequence (.sup.224 Ra, .sup.223 Ra, .sup.225 Ac), 
also with the chance that daughters thereof may redistribute before decay, 
g. the radioactive halflife of some isotopes is so long that most of the 
activity leaves the patient undecayed, resulting in a waste problem 
(.sup.223 Ra, .sup.224 Ra, .sup.225 Ac), or 
h. the halflife of the isotope is so short that most of the isotope decays 
before reaching its ultimate target (.sup.212 Bi, .sup.213 Bi), 
i. sufficient precursor material may not be available to extract at one 
time the necessary amount of isotope for a single patient treatment 
(.sup.212 Bi, .sup.213 Bi), and, 
j. isotopes producing hard gamma rays in their isotope decay need shielding 
facilities to prevent radiation hazards to technicians and nursing 
personnel. 
One or more of the arguments listed above will make it very difficult, if 
not impossible for some of the isotopes to ever be used on a large scale 
for .alpha.-radio-immunotherapy, and that in particular if one or more of 
the others can be used on acceptable technical, logistical and financial 
conditions. 
The closest prior art to the present invention (the French patent 
application FR-A-2 527 928) discloses a method to produce conjugates of an 
antibody and .sup.212 Bi. However, these conjugates still suffer from the 
drawbacks mentioned above under e, h, i and j. 
SUMMARY OF THE INVENTION 
The present invention provides a method for producing a conjugate of a 
targeting moiety and at least one radioisotope at or near the bedside of a 
patient, characterized in that a relatively long lived radioisotope of 
which the daughters in the decay sequence predominantly emit .alpha. 
and/or .beta. rays, is loaded on an appropriate medium out of which medium 
a relatively short lived isotope is eluted and coupled to a targeting 
moiety. 
Relatively long lived in this context means that the radioisotopes have a 
decay time in the order of several days, which enables sufficient time for 
packing and shipping. Relatively short lived in this context mean that the 
radioisotopes have a decay time in the order of minutes or hours. 
With a decay sequence in which predominantly .alpha.- and/or .beta.-rays 
are emitted is meant a decay sequence which does not cause danger for 
radiation hazards caused by gamma rays to people working with the 
compounds without the burden of applying protective shielding.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS 
An important aspect of the present invention is that the radioconjugate can 
be made or quasi be made at the site of therapy. Due to the decay sequence 
which results mainly in .alpha.- and/or .beta.-radiation it has become 
possible that no protection against radiation is necessary. This is 
extremely useful, because due to the absence of gamma radiation it has 
become possible that the conjugation can be done at or near the bed side 
without the necessity to apply radiation shielding or isolation of the 
patient. This is not only preferable from the point of radiation danger 
but is also gives advantages for the availability of the short-living 
isotope. This isotope can be prepared in the neighboorhood of the patient 
allowing for a rapid administration and prevention of loss of therapeutic 
action caused by the rapid decay of the isotope. In this way it has become 
possible to use short lived radioisotopes for therapy. 
An ion exchange column or another appropriate substrate filled with the 
long lived isotope can be placed at or near the bedside, for instance, 
where the short-lived isotope can be eluted by washing the substrate with 
a suitable solution. After elution the short lived isotope is coupled to 
the targeting moiety and (optionally together with an infusion solution) 
the conjugate can be administered. This can all be done in a continuous 
mode with an apparatus according to the invention as shown in FIG. 1 or 
FIG. 3, or in an intermittent mode by using ordinary laboratory glassware. 
Of course it may also be made possible to add the targeting moiety to the 
eluting solution so that the coupling takes place in the column. 
This invention primarily addresses the use of the shortest-lived isotope 
from the list mentioned above, .sup.213 Bi. The invention enables the 
person skilled in the art to milk this isotope by a continuous or an 
intermittent extraction-method from one of its precursors, .sup.225 Ac, at 
the bed-side of the patient, or in the nearestby hospital laboratory 
facility, to link the .sup.213 Bi in a continuous or an intermittent 
manner onto the targeting moiety, to either or not mix the conjugate 
solution with an infusion liquid and to administer this mixture 
intravenously to the patient--for example as is schematically pictured in 
FIG. 1. 
At first sight this procedure might seem extremely wasteful, because 
.sup.225 Ac, itself being an .alpha.-emitting isotope, produces three 
potentially therapeutically useful .alpha.-particles before yielding the 
.sup.213 Bi-isotope, as is shown in FIG. 2. However, the source material 
for .sup.225 Ac, .sup.229 Th, and thereby also the .sup.225 Ac itself, can 
be made available at sufficiently low cost to allow it to be used in the 
proposed manner on economically justifiable terms. 
The use of .sup.213 Bi is not only preferable from a viewpoint of radiation 
hazards. It is also preferable because no gaseous isotopes occur in the 
decay sequence of its precursors. This is advantageous over the use of 
other isotopes which have a decay with a gaseous isotope which 
necessitates the handling and reaction environment to be air-tight. 
Milking, conjugation and administration of .sup.213 Bi are not hampered by 
the necessity for having air-tight conditions and the reactions can be 
done under normal conditions. 
The targeting moiety may preferably be a monoclonal antibody, or a fragment 
or a derivative thereof. Preferably such an antibody is a human or a 
humanized antibody to prevent immunologic reactions to the antibody. 
Non-human antibodies are mostly of murine origin. These, like all other 
foreign proteins, are highly immunogenic in man. The phenomenon of HAMA, 
human anti mouse antibodies, is well known in the field and severely 
limits the use of mouse derived antibodies in diagnostic and especially in 
therapeutic applications in human beings. A single application of a murine 
antibody is usually sufficient to mount an immune response that will 
prevent subsequent applications to be effective. 
Of course fragments and/or derivatives of the targeting moieties can also 
be used, as long as they retain a substantial amount of target 
specificity. Thus, for this invention it should be understood that where a 
targeting moiety is mentioned one should also consider a fragment or a 
derivative thereof as part of the invention. 
Preferably antibodies are directed against tumor associated antigens, such 
as CEA (Carcino-embryonic antigen), AFP (alpha-foetoprotein), FHAP (fast 
homoarginine-sensitive alkaline phosphatase), p97 (melanome specific), and 
EL-1 (elongation factor 1). 
Another preferable targeting moeity is formed by a ligand for a cell 
surface receptor or a fragment or derivative of such a ligand. Examples of 
such ligands are agonists and/or antagonists of pharmacologically active 
receptors, but also T cell epitopes which can bind to the T cell receptor 
are prefered. 
Another aspect of the invention provides a method for treating numerous 
patients with one ion exchange column loaded with isotope. The amount of 
isotope loaded depends on the number of patients to be treated. The wanted 
isotope can be eluted from the column intermittently, with suitable 
intervals depending on the half-lifes in the decay chain. 
With related tumours or infectious organisms the same targeting moiety (or 
mix of targeting moieties) may be used for various patients. For unrelated 
diseases there must be a means for changing the targetting moiety 
preparation. 
The coupling of the isotope to the targeting moiety can be done in any 
suitable way, as long as the targeting specificity of the targeting moiety 
is not hampered to a substantial amount. 
Preferably the coupling will be done through one of the now many known 
chelating agents. As already disclosed, it may be advantageous to couple 
the isotopes to a carrier, such as HSA, which of course can also be done 
through chelating agents. The advantage of a carrier is that a large 
number of radioisotopes can be brought to the target cell. Since it is 
assumed that several .alpha.-particles are necessary for the destruction 
of one target cell an increase in the number of isotopes in the direct 
neighbourhood of the target cell is preferable. 
The invention also provides a conjugate as produced by the method of the 
invention, as well as a pharmaceutical formulation comprising such a 
conjugate. 
A method is provided for producing the conjugate of a targeting moiety and 
a radioisotope and administering it to the patient without delay or any 
necessary actions of the therapist. 
Another aspect of the invention provides an apparatus for carrying out site 
directed therapy or imaging. 
The simplest way to describe the method and apparatus, subject of this 
invention, with reference to FIG. 1, is as follows: 
A capillary column contains, by means of example, twice the amount of 
precursor-.sup.225 Ac needed for a single patient dose of .sup.213 Bi. 
Example: in a case the patient dose corresponds with 30 mCi (equals 
2.10.sup.-9 g) of .sup.213 Bi over a 10 day period, the capillary column 
(3) will contain 200 .mu.Ci of .sup.225 Ac (equals 4.10.sup.-9 g). 
The .sup.225 Ac is present in a 3.sup.+ form on a suitable ionexchange 
substrate. Upon its (continuously occurring) decay it is stripped from the 
column by a certain overdose of the eluent in flask (1) containing the 
appropriate targetting moiety capable of binding the isotope. The binding 
part of the targetting moiety and other chemical equilibrium conditions of 
the eluent-ionexchange system are chosen such that the .sup.213 Bi, for 
all practical purposes, quantitatively binds to the targetting moiety. The 
immediate daughter of .sup.225 Ac, .sup.221 Fr has a radioactive decay 
halflife of 4.8 minutes. It is this isotope which acts via the very 
short-lived .sup.217 At as the direct precursor of .sup.113 Bi. In case 
the .sup.221 Fr is not retained by itself or in the ion exchange 
substrate, the delaying effect of the .sup.221 Fr-halflife causes the need 
of a certain period of time between the decay of .sup.225 Ac at and its 
stripping from the capillary column and the binding of the .sup.213 Bi 
onto the targetting moieties. The optimum value for such a delay is 
somewhere between the halflife of the .sup.221 Fr and the halflife of the 
.sup.213 Bi isotopes. 
This delay can be effected by the length of tubing between the capillary 
(3) and the patient (4), if necessary enhanced by an extra length of 
intermediate tubing, as indicated in FIG. 1 as (5). The infusion liquid 
from flask (2) enters the patient, it is mixed with the isotope-containing 
eluate from column (3), as indicated as junction (6) in FIG. 1. 
In order to obtain optimal stripping and conjugation conditions in the 
capillary column (3), it may be that the composition of the eluent in 
flask (1) is not optimal (for example its pH-value) for administration to 
the patient. Presuming that the volume rate of infusion liquid is an order 
of magnitude higher than of the eluate liquid, this can easily be 
countered for by a compensating off-balance (buffered) pH-value of the 
infusion liquid. 
It is also possible that the binding of the targeting moiety is hampered by 
the physico-chemical properties of the eluent. Therefore, an other 
embodiment of the invention is represented in FIG. 3 where an eluens is 
lead from a vessel (7) through an ion exchange column (3) so that a 
radioisotope is stripped from that column. The eluens containing the 
isotope is mixed with a liquid from a vessel (1) containing a targeting 
moiety, so that the isotope is bound to the targeting moiety. The 
resulting fluid is mixed with infusion liquid from vessel (2) at junction 
(6) and administered to the patient (4). Optionally the eluens containing 
the isotope may be lead through an additional length of tubing (5) to 
correct for the half-life of intermediate daughter isotopes. 
What the invention enables in terms of the development and the clinical use 
of .alpha.-radioimmunotherapy, in this case using .sup.213 Bi as the 
active cell-killing agent is: 
"single patient kits" in the form of precursor with a halflife that is 
logistically managable regarding: 
minimization of active material loss by radioactive decay during operations 
like packaging, transport, etc., 
safety in transportation over long distances and in handling in hospitals, 
applicability in practice on a large scale in many hospitals without need 
for special precautions, regarding: 
the handling of the material and the application procedures regarding the 
treatment of patients, all without complicated monitoring equipment, 
collection and handling facilities for (urinous) waste, 
maximal (and in case of continous extraction, almost total) use of the 
.sup.213 Bi after it is generated from the precursor isotope, 
maximum flexibility in dose administration by the possibility of changing 
treatment time, allowing for a minimum range of single patient kit 
precursor concentration standards. 
All these aspects then pertain precisely to fields where the short-range 
.alpha.-particles are most suited for their potential therapeutic uses 
like: 
micrometastases (of less than 1 mm diameter) of various cancers, 
cellular cancers like leukemias and 
also, certain kinds of very localized autoimmune diseases, all of which can 
essentially be directly addressed either by the blood-circulation system 
or locally without the need for slow diffusion processes of the 
antibody-ligand-isotope complexes through intercellular space in order to 
find their ultimate destination. 
A special advantage of intermittent administration of the therapeutic 
radioconjugates is the advantage which occurs by dose fractionation. 
Statistically it is possible to calculate the dose needed to kill 99.9% of 
the tumor cells with a dose of radioconjugate: assuming that a leukemic 
(monocellular, blood and marrow bone) tumor load of 1 kg exisits, which is 
roughly equal to 10.sup.12 cells, and that 10 .alpha.-particles are needed 
to kill a cell (6 MeV), then 10.sup.13 .alpha.-particles would be needed, 
which corresponds with 50 mCi .sup.213 Bi. Thus for a single dose, which 
would kill 99.9% of the tumor cells 50 mCi .sup.213 Bi would be needed. 
The "dose versus survival" relation for this cell morphology with 6 MeV 
.alpha.-particles can be derived from the formula D/D.sub.0 =-ln S, in 
which S=survival fraction, D=dose administered and D.sub.0 =reference dose 
for 37% survival. From this formula the following Table of values can be 
calculated: 
TABLE 1 
______________________________________ 
Dose versus kill ratio for tumor cells. The numbers are the 
number of .alpha.-particles necessary to kill the given % of tumor 
cells. 
In case A 600 rad are necessary to obtain a 99% kill ratio. In case 
B 2000 rad is assumed necessary for the same effect. 
Cell kill in % Case A Case B 
______________________________________ 
0 0 0 
1 0.015 0.05 
10 0.15 0.5 
40 0.7 2 
50 1 3 
60 1.3 4 
70 1.5 5 
90 3 10 
99 6 20 
99.9 9 30 
99.99 12 40 
99.999 15 50 
______________________________________ 
From this Table the effects of an intermittent, dose fractioned, 
administration can be read: 
The effect of cell survival of successive doses of 5 mCi .sup.213 Bi in 
case A is as follows: 
the first dose of 5 mCi equals 1 .alpha.-particle per cell, which gives 50% 
survival, which means that 0.5.multidot.10.sup.12 cells remain; 
the second dose of 5 mCi equals 2 .alpha.-particles per cell, which gives 
20% survival, which means that 0.1.multidot.10.sup.12 cells remain; 
the third dose of 5 mCi equals 10 .alpha.-particles per cell, which gives 
0.1% survival, which means that 0.1.multidot.10.sup.9 cells remain; 
the fourth dose of 5 mCi equals 10,000 .alpha.-particles per cell, which 
means a total kill. 
Thus it can be shown that by intermittent dosing a total dose of 4 times 
5=20 mCi .sup.213 Bi is sufficient to give a total kill of the tumor 
cells. For clarity the effects of intermediate tumor growth and 
maximization of the number of targeting moieties on the tumor cells have 
been omitted. Nevertheless, it is clear that by intermittent 
administration the total load of radioactive material can be kept smaller. 
Even in case B, which has a more unfavourable dose versus survival rate, 
and advantageous effect is realized: 
EQU 1.sup.st dose.fwdarw.1.alpha./cell.fwdarw.75% 
survival.fwdarw.0.75.multidot.10.sup.12 cells 
EQU 2.sup.nd dose.fwdarw.1.3.alpha./cell.fwdarw.70% 
survival.fwdarw.0.50.multidot.10.sup.12 cells 
EQU 3.sup.rd dose.fwdarw.2.alpha./cell.fwdarw.60% 
survival.fwdarw.0.30.multidot.10.sup.12 cells 
EQU 4.sup.th dose.fwdarw.3.alpha./cell.fwdarw.50% 
survival.fwdarw.0.15.multidot.10.sup.12 cells 
EQU 5.sup.th dose.fwdarw.6.alpha./cell.fwdarw.25% 
survival.fwdarw.0.04.multidot.10.sup.12 cells 
EQU 6.sup.th dose.fwdarw.25.alpha./cell.fwdarw.0.3% 
survival.fwdarw.0.1.multidot.10.sup.9 cells 
EQU 7.sup.th dose.fwdarw.10,000.alpha./cell.fwdarw.total kill after 35 mCi. 
There are two ways presently known to obtain .sup.229 Th as a precursor for 
the .sup.225 Ac-source-isotope: 
from stockpiled .sup.233 U, by its natural .alpha.-decay. Batches of 
.sup.233 U were made in nuclear breeder reactors about 30 years ago, but 
never used as nuclear fuel. 
Some of the .sup.233 U was separated from the bulk-.sup.233 Th, from which 
it was made, so that the now available .sup.229 Th can be obtained in 
highly pure form. 
by high neutron flux irradiation from natural .sup.226 Ra, with .sup.227 Ac 
as an intermediate product. Futher irradiation of this .sup.227 Ac yields 
roughly equal amounts of .sup.229 Th and .sup.228 Th, the latter with much 
shorter halflife (2 years) than the .sup.229 Th. On the one hand this 
complicates the extraction of .sup.225 Ac considerably, but in properly 
equipped installations it may on the other hand yield .sup.224 Ra, an 
.alpha.-emitter with a 3.7 day halflife. When the Ra is properly isolated, 
it may be used as a source for .sup.212 pb. 
The 10.5 hour halflife of .sup.212 Pb will cause considerable complications 
in handling. However, when these are properly taken care of, one may 
envisage to use the .sup.212 Pb-isotope in the same manner as the .sup.225 
Ac in this invention as a bed-side source of .sup.212 Bi, which for al 
practicle purposes acts as an .alpha.-emitter with a halflife of 1.0 hour. 
EXAMPLES 
Example 1 
The separation chemistry of the various radioactive elements mentioned in 
the text before has been sorted out decades ago and is well-documented in 
the public literature. Examples are references (3) and (4). .sup.225 Ac 
can be separated from .sup.229 Th on a Dowex 50 ionexchanger by stripping 
with 4N HNO.sub.3. After evaporation of the acid, the .sup.225 Ac can be 
dissolved again in 0.5N HNO.sub.3 in a fixed concentration and absorbed in 
the appropriate amount on Dowex 50, which then becomes the material in the 
mini-column (3) of FIG. 3. 
Example 2 
0.68.+-.0.07 mCi of .sup.225 Ac was obtained from the European Joint 
Research Centre. This was loaded on a MP-50 cation exchange resin 
(Bio-Rad). The formed .sup.213 Bi was eluted with a mixture of 50:50 10% 
NH.sub.4 Ac:MeOH with a pH of 6.75. An autoburet was used to deliver 35 
.mu.l of eluant per minute; alternatively, manual elution was done at 50 
.mu.l amounts of eluant per minute. 
In a few experiments it was necessary to purify the .sup.213 Bi. This was 
accomplished by heating the eluant to dryness in a 10 ml beaker containing 
0.5 ml of conc. HNO.sub.3. After evaporation under an IR lamp, the bismuth 
activity was transferred to a column of MP-50 resin (2.times.30 cm, 
pre-equilibrated with 0.1M HNO.sub.3). The resin was washed with 0.2 ml 
H.sub.2 O. Then the .sup.213 Bi was eluted with 0.5 ml of HCl and HI. 
Various concentrations of HCl and HI have been tried. FIG. 4 shows the 
elution patterns for .sup.213 Bi. In all cases the elution is rapid and 
quantitative. All of the isotope can be obtained within 5 to 10 minutes 
after the start of the elution. 
Example 3 
Radiolabeling was done by adding enough 3M NH.sub.4 Ac to the .sup.213 Bi 
stock to achieve pH 4.0-5.0. Then 53 .mu.l or 106 .mu.l of a 4.7 mg/ml 
solution of monoclonal antibody B3 coupled with the chelator CHX-DTPA 
(cyclohexyldiethylenetriaminepenta acetic acid) according to the method 
described in (5) were gently mixed into the solution. After a fifteen 
minute reaction time, 1.5 .mu.l of 0.1M EDTA were added. The solution was 
transferred to a 1 ml syringe with 0.2 ml wash. The solution was then 
injected into the HPLC (high pressure liquid chromatography) having a TSK 
3000 column. The buffer was 0.02M MES/Cl.sup.- (MES=morpholino ethane 
sulfonic acid), 0.15M NaCl, pH 6.5. Elution of the B3 antibody occurred at 
7.5 minutes. The amount of .sup.213 Bi incorporated into the antibody was 
monitored with an in-line radiochemical detector (Beckman). All activity 
measurements of .sup.213 Bi were corrected for decay (t.sub.1/2 =45.6 
min). Results are depicted in Table 2. Activities of .sup.225 Ac, .sup.221 
Fr or .sup.217 At were not detectable in any of the .sup.213 Bi elution 
products. 
TABLE 2 
______________________________________ 
Results of radiolabeling experiments incorporating 
.sup.213 Bi into mAb B3-CHX-DTPA. 
.sup.213 Bi 
Acid Vol. acid .mu.l 
.mu.g mAb recovered in mAb (%) 
______________________________________ 
2M HCl 210 250 43.4 (37%) 
2M HCl 210 500 45.0 (25%) 
0.1M HCl 
500 500 3.4 (7%) 
0.1M HI 
______________________________________ 
Example 4 
In the same way as described in Example 2 and 3, .sup.213 Bi was eluted 
from .sup.225 Ac and coupled to a targeting moiety. For this experiment a 
conjugate of monoclonal antibody M195 and the chelator CHX-DTPA was used. 
Table 3 summarizes the results. 
TABLE 3 
______________________________________ 
Results of radiolabeling experiments incorporating 
.sup.213 Bi into mAb M195-CHX-DTPA. 
.mu.g antibody 
.sup.213 Bi recovered (%) 
______________________________________ 
50 198 (53%) 
25 107 (39%) 
______________________________________ 
REFERENCES 
(1) D. R. Fisher: ".alpha.-particle emmitters in medicine", proceedings of 
a symposium held at Loews L'Enfant Plaze Hotel, Washington, D.C., 
September 21 and 22, 1989, pages 194-214, published by the American 
College of nuclear physicians. 
(2) D. S. Wilbur: "Potential use of .alpha.-emitting radionuclides in the 
treatment of cancer", Antibody, Immunoconjugates, and 
Radiopharmaceuticals, volume 4, number 1, 1991, pages 85-97, published by 
Mary Ann Liebert, Inc. 
(3) T. Mitsugashira: "Preparation of traces for actinium, thorium, 
protactinium and uranium", SPEY, Min. Educ. Sci. & Cult., Tokyo, 9, 1984, 
pages 111-116. 
(4) S. Suzuki: "Solution chemistry of light actinide elements", Japan-US 
seminar on Thorium fuel reactors--proceedings, Nara, Japan, 18-22 October 
1982 (Tokyo, 1985) pages 137-143. 
(5) Mirzadeh, S., Brechbiel, M. W., Atcher, R. W., Gansow, O. A., 
Bioconjugate Chem., volume 1, 1990, 59-65.