Methods for technetium/rhenium labeling of proteins

A method for radiolabeling a protein with a radioisotope of technetium or rhenium is disclosed which comprises the steps of contacting a solution of a protein containing a plurality of adjacent free sulfhydryl groups, or in particular cases, intact protein containing at least one disulfide group, with stannous ions, and then with radiopertechnetate or radioperrhenate, the amount of stannous ion being sufficient to substantially completely reduce the radiopertechnetate or radioperrhenate, and recovering radiolabeled protein. A rapid and quantitative method for producing a sterile, injectable solution of Tc-99m-labeled monovalent antibody fragment is also disclosed which comprises the step of mixing a sterile solution containing a monovalent antibody fragment having at least one free sulfhydryl group, stannous chloride and excess tartrate, at mildly acidic pH, or a sucrose-stabilized lyophilizate of such solution, with a sterile solution of Tc-99m-pertechnetate, whereby substantially quantitative labeling of the antibody fragment with Tc-99m is effected in about 5 minutes at ambient temperature, the resultant sterile solution of Tc-99m-labeled monovalent antibody fragment being suitable for immediate injection into a patient for scintigraphic imaging.

The present invention relates to improved and optimized methods for direct 
labeling of proteins, especially antibodies and/or antibody fragments, 
with radioisotopes of technetium and rhenium. 
The present further invention relates to a method and kit for directly and 
rapidly radiolabeling a monovalent antibody fragment with technetium-99m 
(Tc-99m), using one or more pendant sulfhydryl groups as endogenous 
ligands, and more particularly to a method and kit for radiolabeling Fab 
or Fab' antibody fragments to prepare a sterile, Tc-99m-labeled antibody 
fragment solution which is almost immediately ready for injection into a 
patient for scintigraphic imaging. 
The isotope technetium-99m is among the most valuable in diagnostic nuclear 
medicine due to its ready availability, low cost and favorable 
radiochemical characteristics. It is used widely as an agent for labeling 
macromolecules such as monoclonal antibodies and can be bound to the 
protein in various ways. Early work mainly used the bifunctional chelate 
approach, i.e., use of a chelator which contained another functional group 
for linkage to the protein. Various forms of diethylenetriaminepentaacetic 
acid (DTPA) were used, for example, to bind to the antibody and also to 
chelate the radiometal ion. 
Direct labeling of protein was also tried, using a "pretinning" protocol, 
requiring severe conditions and long "pretinning" times, but radiolabeling 
at 100% incorporation was not achieved. Moreover, the presence of 
extremely high amounts of stannous ion for long periods compromised the 
immunoreactivity of the antibody. The process also generally necessitated 
a post-labeling purification column. Attempts to repeat pretinning 
procedures of others with F(ab').sub.2 antibody fragments were 
unsatisfactory in achieving Tc-99m labeling. 
Other, more recent direct labeling methods have required separate vials, 
one for antibody and one for stannous ion complexed to a transchelator 
such as a phosphate and/or phosphonate. 
European Patent Application A2/0 237 150, to NeoRx Corp., and PCT 
Application WO 88/07382, to Centocor Cardiovascular Imaging Partners, 
L.P., each disclose methods for radiolabeling an antibody or antibody 
fragment with Tc-99m, but the labeling conditions are not optimized for 
labeling Fab or Fab' fragments and the disclosed conditions are 
inconvenient and do not result in quantitative labeling. 
The element below technetium in the periodic table, rhenium, has similar 
chemical properties and might be expected to react in an analogous manner 
to technetium. There are some 34 isotopes of rhenium and two of them in 
particular, rhenium-186 (t 1/2, 90h; gamma 137 keV, beta 1.07, 0.93 MeV) 
and rhenium-188 (t 1/2, 17h; gamma 155 keV, beta 2.12 MeV), are prime 
candidates for radioimmunotherapy using monoclonal antibody approaches. 
Both isotopes also have gamma emmissions at suitable energies for gamma 
camera imaging purposes. Rhenium-186 is obtained from reactor facilities 
by bombardment of enriched rhenium-185 with neutrons, which yields 
rhenium-186 in a "carrier-added" form containing a large excess of 
non-radioactive rhenium-185. Rhenium-188 is obtained from a 
tungsten-188/rhenium-188 generator (Oak Ridge National Laboratory) and can 
be eluted from the generator in a substantially carrier-free form with 
little tungsten breakthrough. Also, the energy deposition from this 
isotope at a high .DELTA.=1.63 g-rad/.mu.Ci-h is close to another potently 
energetic potential therapeutic, yttrium-90 (.DELTA.=1.99 g-rad/.mu.Ci-h) 
while at the same time the chemical properties of rhenium may make it less 
of a bone-seeking agent than yttrium (which is often contaminated with 
strontium-90) and give rise to better tumor/organ biodistribution and 
dosimetry. 
Although many groups have alluded to the possibility of utilizing rhenium 
to label antibodies in the same fashion as technetium, little successful 
work has been published. Low rhenium incorporations are usually seen with 
antibody-chelate conjugates and there is a general tendency of rhenium to 
reoxidize back to perrhenate and then dissociate from complexation. 
Besides, use of the bifunctional chelate approach often requires an 
organic synthesis with a lengthy series of intermediates to be isolated 
and purified prior to antibody conjugation. 
A need continues to exist for a simple, one-vial method for radiolabeling 
proteins with radioisotopes of technetium and rhenium. 
A need also continues to exist for a direct method for stably radiolabeling 
Fab and Fab' antibody fragments with Tc-99m within a few minutes to 
produce an solution which is ready for immediate injection into a patient 
for scintigraphic imaging. 
OBJECTS OF THE INVENTION 
One object of the present invention is to readily produce a highly 
immunoreactive technetium or rhenium radiolabeled antibody or antibody 
fragment which is stable to loss of label by transchelation or 
reoxidation. 
Another object of the invention is to provide a method for direct 
radiolabeling of a protein which produces high yields of labeled product 
with minimal contamination with by-products. 
Another object of the invention is to provide a convenient and efficient 
radiolabeling kit for use in introducing technetium or rhenium 
radioisotope into an antibody or antibody fragment. 
Another object of the present invention to provide a method for direct 
Tc-99m radiolabeling of a monovalent, e,g,. Fab or Fab', antibody fragment 
which is rapid and convenient and which results in a labeled fragment 
ready for direct injection into a patient. 
Another object of the invention is to provide an "instant" Tc-99m labeling 
kit for labeling a Fab or Fab' antibody fragment that is stable to 
prolonged storage but that can be combined directly with the sterile 
saline effluent from a Tc-99m generator to produce a sterile solution of 
radioantibody fragment. 
Upon further study of the specification and appended claims, further 
objects and advantages of this invention will become apparent to those 
skilled in the art. 
SUMMARY OF THE INVENTION 
The foregoing objectives are achieved, according to one aspect of the 
invention, by providing a method for radiolabeling a protein containing a 
plurality of spatially adjacent free sulfhydryl groups with a radioisotope 
of technetium or rhenium, comprising the steps of contacting a solution of 
said protein containing a plurality of spatially adjacent free sulfhydryl 
groups with stannous ions, and then with radiopertechnetate or 
radioperrhenate, the amount of stannous ion being in slight excess in the 
case of technetium and in greater excess in the case of rhenium over that 
required to substantially completely reduce said radiopertechnetate or 
radioperrhenate, and recovering radiolabeled protein. 
According to another aspect of the invention, there is provided a method 
for producing a sterile, injectable solution of Tc-99m-labeled monovalent 
antibody fragment, which comprises the step of mixing: 
(1A) a sterile solution containing a unit dose for scintigraphic imaging of 
a monovalent antibody fragment having at least one free sulfhydryl group, 
stannous chloride in an amount of about 100-150 .mu.g Sn per mg of 
antibody fragment, and about a 30-40-fold molar excess of tartrate over 
stannous chloride, in about 0.04-0.06 M acetate buffer containing saline, 
at a pH of 4.5-5.0, or 
(1B) the lyophilizate of a sterile solution containing a unit dose for 
scintigraphic imaging of a monovalent antibody fragment having at least 
one free sulfhydryl group, stannous chloride in an amount of about 100-150 
.mu.g Sn per mg of antibody fragment, and about a 30-40-fold molar excess 
of tartrate over pH of 4.5-5.0, stannous chloride, in about 0.04-0.06 M 
acetate buffer containing saline and made about 0.08-0.1 M in sucrose, at 
a pH of 4.5-5.0, 
with 
(2) a sterile solution containing an effective scintigraphic imaging amount 
of Tc-99m-pertechnetate, 
whereby substantially quantitative labeling of the antibody fragment with 
Tc-99m is effected in about 5 minutes at ambient temperature, the 
resultant sterile solution of Tc-99m-labeled monovalent antibody fragment 
being suitable for immediate injection into a patient for scintigraphic 
imaging. 
The invention also provides technetium or rhenium radio-labeling kits for 
effecting the labeling process of the invention, especially for producing 
Tc-99m and rhenium radiolabeled antibodies and antibody fragments, 
processes for their production, and improved methods of radioantibody 
imaging and therapy using antibodies and antibody fragments radioalabeled 
according to the invention.

DETAILED DESCRIPTION 
It has now been found that a protein, in particular an antibody or antibody 
fragment, having a plurality of spatially adjacent free sulfhydryl groups 
can selectively bind technetium and rhenium radiometal ions, under mild 
conditions, to form tight bonds to the sulfhydryl groups that are quite 
stable in blood and other bodily fluids and tissues. Both the reagents and 
the conditions in the present method are greatly simplified, but the 
method is particularly adapted for technetium or rhenium labeling and it 
is surprisingly and unexpectedly shown that optimal conditions for each 
label are different. 
A first method according to the invention thus broadly comprises the step 
of contacting a solution of a protein containing a plurality of spatially 
adjacent free sulfhydryl groups, said solution also containing tin(II) 
ions, with a solution of Tc-99m-pertechnetate or perrhenate (using a 
radioisotope of rhenium of therapeutic or imaging utility) ions, whereby a 
solution of technetium or rhenium radiolabeled protein is obtained. The 
procedure is simple and practical for the nuclear medicine physician and 
technologist. Preferred embodiments of the first method include applying 
the method of the invention to produce radiolabeled antibodies or antibody 
fragments useful for gamma imaging and radioisotope therapy. 
The first labeling method and kit of the invention may be used to bind 
radioiosotopes of technetium and rhenium to other proteins with the 
requisite free sulfhydryl groups. Proteins which contain two or more 
proximal free sulfhydryl groups can be labeled directly. Those which 
contain disulfide groups, normally linked through a cystine residue, can 
be treated with a reducing agent to generate the free sulfhydryl groups. 
This may result in fragmentation of the protein if the disulfide bond 
links polypeptide chains which are not continuous, or it may merely result 
in chain separation, possibly involving a change in conformation of the 
protein if the disulfide bond joins remote segments of a single 
polypeptide chain. Sulfhydryl groups can be introduced into a polypeptide 
chain to provide the requisite proximal groups. 
Reduction of an antibody or F(ab').sub.2 fragment with known disulfide bond 
reducing agents, e.g., dithiothreitol, cysteine, mercaptoethanol and the 
like, gives after a short time, typically less than one hour, including 
purification, antibody having from 1-10 free sulfhydryl groups by 
analysis. When labeled with technetium using a reducing agent such as 
stannous ion under the present conditions, 100% incorporation of Tc-99m to 
protein is seen together with &gt;95% retention of immunoreactivity. 
The methods of the invention are particularly attractive for labeling 
antibodies and antibody fragments, although proteins such as albumin, 
drugs, cytokines, enzymes, hormones, immune modulators, receptor proteins 
and the like may also be labeled. Antibodies contain one or more disulfide 
bonds which link the heavy chains, as well as disulfide bonds which join 
light and heavy chains together. The latter disulfide bonds are normally 
less accessible to disulfide reducing agents and the bonds linking heavy 
chains can normally be selectively cleaved. The resultant fragments retain 
their immunospecificity and ability to bind to antigen. It will be 
understood that reduction of disulfide bonds linking the heavy chains of 
an immunoglobulin must be effected with care, since the normally less 
reactive disulfide bonds linking light and heavy chains will eventually be 
reduced if reducing conditions are too drastic or the reducing agent is 
left in contact with the fragments for too long a time. 
Once reduced, the antibody-SH moleties are quite stable if stored under 
rigorously oxygen-free conditions. Stability is also increased with 
storage at lower pH, particularly below pH 6. 
It will also be understood that the antibodies or antibody fragments to be 
radiolabeled can be antibodies or fragments thereof which bind to antigens 
which include but are not limited to antigens produced by or associated 
with tumors, infectious lesions, microorganisms, parasites, myocardial 
infarctions, clots, atherosclerotic plaque, or normal organs or tissues. 
By "antibodies and antibody fragments" is meant generally immunoglobulins 
that specifically bind to antigens to form immune complexes. The terms 
include conventional IgG, IgA, IgE, IgM, and the like, conventional enzyme 
digestion products such as F(ab').sub.2 fragments obtained by pepsin 
digestion of intact immunoglobulins, Fab fragments obtained by papain 
digestion of intact immunoglobulins, conventional monovalent Fab' and 
light-heavy chain fragments obtained by disulfide bond cleavage of 
F(ab').sub.2 fragments and intact antibody, respectively. Cleavage is 
advantageously effected with thiol reducing agents, e.g., cysteine, 
mercaptoethanol, dithiothreitol (DTT), glutathione and the like. However, 
monovalent fragments can also include any fragments retaining the 
hypervariable, antigen-binding region of an immunoglobulin and having a 
size similar to or smaller than a Fab' fragment. Products having 
substantially similar properties to such immunoglobulins and fragments are 
also included. Such similar proteins include antibody subfragments made by 
further digestion or manipulation of larger fragments, genetically 
engineered antibodies and/or fragments, whether single-chain or 
multiple-chain, and synthetic proteins having an antigen recognition 
domain which specifically binds to an antigen and otherwise functions in 
vivo in a substantially analogous fashion to a "classical" immunoglobulin. 
The only substantive requirement for such a protein to be useful in the 
first method according to the invention is that it have two or more 
proximal sulfhydryl groups to serve as chelators for the reduced 
pertechnetate or reduced perrhenate radiometal ion. 
The cleaved F(ab').sub.2 fragment containing at least one free sulfhydryl 
group, which is useful in a second method according to the invention, will 
be termed "Fab'-SH" herein. Cleaved F(ab).sub.2 will be termed "Fab-SH" 
herein. 
Reduction of F(ab').sub.2 fragments is preferably effected at pH 5.5-7.5, 
preferably 6.0-7.0, more preferably 6.4-6.8, and most preferably at about 
pH 6.6, e.g., in citrate, acetate or phosphate buffer, preferably 
phosphate-buffered saline, and advantageously under an inert gas 
atmosphere. It is well known that thiol reduction can result in chain 
separation of the light and heavy chains of the fragment if care is not 
taken, and the reaction must be carefully controlled to avoid loss of 
integrity of the fragment. 
Cysteine is preferred for such disulfide reductions and other thiols with 
similar oxidation potentials to cysteine will also be advantageously used. 
The ratio of disulfide reducing agent to protein is a function of 
interchain disulfide bond stabilities and must be optimized for each 
individual case. Cleavage of F(ab').sub.2 antibody fragments is 
advantageously effected with 10-30 mM cysteine, preferably about 20 mM, 
and a protein concentration of about 10 mg/ml. 
Reduction of a F(ab').sub.2 fragment with known disulfide bond reducing 
agents gives after a short time, typically less than one hour, including 
purification, Fab' typically having 1-3 free sulfhydryl groups by 
analysis. Sulfhydryl groups can be introduced into an antibody fragment to 
improve radiometal binding. Use of Traut's Reagent (iminothiolane) for 
this purpose is not preferred, whereas use of oligopeptides containing 
several adjacent sulfhydryl groups is efficacious. In particular, use of 
metallothionein or, preferably, its C-terminal hexapeptide fragment 
(hereinafter, "MCTP"), is advantageous. 
The Fab-SH or Fab'-SH fragments are advantageously then passed through a 
short sizing gel column which will trap low molecular weight species, 
including excess reducing agent. Suitable such sizing gel columns include, 
e.g., dextrans such as Sephadex G-25, G-50 (Pharmacia), Fractogel TSK HW55 
(EM Science), polyacrylamides such as P-4, P-6 (BioRad), and the like. 
Cleavage can be monitored by, e.g., size exclusion HPLC, to adjust 
conditions so that Fab or Fab' fragments are produced to an optimum 
extent, while minimizing light-heavy chain cleavage, which is generally 
less susceptible to disulfide cleavage. 
The eluate from the sizing gel column is then stabilized in about 
0.03-0.07, preferably about 0.05 M acetate buffer, pH about 4.5, made in 
about 0.1-0.3, preferably about 0.15 M saline, and preferably purged with 
an inert gas, e.g. argon. In general, it is advantageous to work with a 
concentration of antibody fragment of about 0.5-5 mg per ml, preferably 
about 1-3 mg/ml, of solution. 
Much less tin(II) is needed to achieve 100% Tc-99m incorporation than was 
previously thought. The general amount of tin used for labeling compounds 
with Tc in most prior art methods is about 100-200 micrograms per 
milligram protein. However, because of the great binding power of the 
sterically close SH groups in the first method of the invention and the 
subnanogram quantities of TcO.sub.4 that normally must be reduced to 
obtain adequate activity for gamma imaging, much less tin(II) can be 
effectively used. Reduction is effected by stannous ion, generally in 
aqueous solution. It will be appreciated that stannous ion is readily 
available as its dihydrate, and also can be generated in situ from tin 
metal, e.g., foil, granules, powder, turnings and the like, by contact 
with aqueous acid, e.g., HCl, and is usually added in the form of 
SnC.sub.12, advantageously in a solution that is also about 0.1 mM in HCl. 
In general, in the first method of the invention it is advantageous to work 
with a concentration of antibody or antibody fragment of about 0.01-10 mg 
per ml, preferably about 0.1-5 mg/ml, of solution, generally in saline, 
preferably buffered to a mildly acidic pH of about 4.0-4.5. In such case, 
the amount of stannous ion needed for reduction of a normal imaging 
activity of pertechnetate is about 0.1-50 .mu.g/ml, preferably about 
0.5-25 .mu.g/ml, in proportion to the amount of protein. 
When labeling the foregoing quantity of antibody or antibody fragment, the 
amount of pertechnetate is generally about 2-50 mCi/mg of antibody or 
antibody fragment, and the time of reaction is about 0.1-10 min. With the 
preferred concentrations of protein and stannous ions noted above, the it 
amount of pertechnetate is preferably about 5-30 mCi/mg, and the time of 
reaction is preferably about 1-5 min. 
Pertechnetate is generally obtained from a commercially available 
generator, most commonly in the form of NaTcO.sub.4, normally in saline 
solution. Other forms of pertechnetate may be used, with appropriate 
modification of the procedure, as would be suggested by the supplier of a 
new form of generator or as would be apparent to the ordinary skilled 
artisan. Pertechnetate is generally used at an activity of about 0 2-10 
mCi/ml in saline, e.g., 0.9% ("physiological") saline, buffered at a pH of 
about 3-7, preferably 3.5-5.5, more preferably about 4.5-5.0. Suitable 
buffers include, e.g., acetate, tartrate, citrate, phosphate and the like. 
The reduction is normally effected under an inert gas atmosphere, e.g., 
nitrogen, argon or the like. The reaction temperature is generally 
maintained at about room temperature, e.g., 18.degree.-25.degree. C. 
These conditions routinely result in substantantially quantitative 
incorporation of the label into the protein in a form which is highly 
stable to oxidation and resistant to transchelation in saline and serum. 
For example, it is now possible to consistently label IgG, previously 
reduced with a thiol-generating reagent, with from 0.5 to 5 micrograms of 
Sn(II) per milligram of IgG, in essentially quantitative yield. Generally, 
at least about 95% of the label remains bound to protein after standing 
overnight at 37.degree. C. in serum. Furthermore the immunoreactivity of 
this protein is hardly reduced after this serum incubation, showing that 
the conjugates are still completely viable imaging agents out to at least 
24 hours. 
At these concentrations, no transchelator such as phosphonate, tartrate, 
glucoheptonate or other well known Sn(II) chelating agent is required to 
keep the tin in solution. Sn(II) compounds such as stannous chloride and 
stannous acetate have been used successfully in these experiments. Other 
readily available and conventional Sn(II) salts are also effective. There 
are only three essential ingredients; the reduced antibody, the aqueous 
stannous ion and the pertechnetate solution. 
Under the conditions described hereunder, 100% of Tc-99m incorporation into 
intact antibody and Fab/Fab' fragments can be readily achieved. In the 
case of F(ab').sub.2, the labeling conditions result in 100% incorporation 
of Tc-99m, but also produce a certain amount of radiolabeled Fab' in 
addition to radiolabeled F(ab').sub.2. 
The resultant Tc-99m-radiolabeled antibodies and antibody fragments are 
suitable for use in scintigraphic imaging of, e.g., tumors, infectious 
lesions, microorganisms, clots, myocardial infarctions, atherosclerotic 
plaque, or normal organs and tissues. Such imaging methods are well known 
in the art. The radioantibody solutions as prepared above are ready at for 
immediate injection, if done in a properly sterilized, pyrogen-free vial. 
Also, no blocking of free sulfhydryl groups after technetium binding is 
necessary for stabilization. 
By "reduced pertechnate" or "reduced perrhenate" is meant the species of 
technetium or rhenium ion formed by stannous ion reduction of 
pertechnetate or perrhenate and chelated by the thiol group(s). It is 
generally thought that reduced pertechnetate is in the form of Tc(III) 
and/or Tc(IV) and/or Tc(V) in such chelates, and that reduced perrhenate 
is in the form of Re(III) and/or Re(IV) and/or Re(V), but higher or lower 
oxidation states and/or multiple oxidation states cannot be excluded and 
are within the scope of the invention. 
Rhenium is found just below technetium in the periodic table, has the same 
outer shell electronic configuration, and therefore might be expected to 
have very similar chemical properties, especially the behavior of 
analogous compounds. In fact, rhenium compounds qualitatively behave 
similarly to technetium compounds insofar as reduction and chelation are 
concerned but their reaction rates are quite different and they are 
dissimilar in certain important respects. 
The radioisotope Re-186 is attractive for therapy and can also be used for 
imaging. It has a half-life of about 3.7 days, a high LET beta emission 
(1.07 MeV) and a convenient gamma emission energy (0.137 MeV). By analogy 
to technetium, rhenium is produced from perrhenate, and the reduced 
rhenium ions can bind nonspecifically to protein. Accordingly, a method 
for Re-186 labeling of proteins, wherein the reduced perrhenate is bound 
to sulfhydryl groups of a protein molecule such as an antibody, would be 
advantageous. Re-188 is a generator-produced beta and gamma emitter with a 
half-life of about 17 hours and is suitable for imaging and therapy. The 
development of commercial generators for rhenium-188 is currently 
underway; and in a preferred scenario, carrier-free rhenium-188 is added 
directly to a vial containing stannous ion and IgG, to produce a rhenium 
radiolabeled protein which is ready for use in less than two hours. The 
half-life of rhenium-188, at 17 hours, makes speed of preparation 
particularly important. 
The procedure of the first method of the invention is modified somewhat in 
the case of rhenium from that used with pertechnetate. In contrast to 
Tc-99m labeling procedures, perrhenate does not label reduced antibodies 
when low amounts of stannous ion reducing agent and short reactions times 
are used. However, by the use of higher amounts of stannous ion, e.g., 
stannous tartrate, and longer reaction times, thiol-reduced antibodies are 
successfully labeled with rhenium. 
By judicious manipulation of conditions, better than 80% rhenium 
incorporations into antibody can be achieved in just a few hours, which is 
particularly important for rhenium-188 with its 17 hour half-life and 
potential for radiobiologic damage to the antibody. Labeling procedures 
are simpler than those currently required for iodine-131 radiolabeling and 
much simpler than what is currently required for labeling antibodies with 
yttrium-90 and copper-67. The short labeling time ensures retention of 
antibody immunoreactivity. 
Conditions will vary depending upon whether the perrhenate is substantially 
carrier-free (e.g., generator-produced Re-188) or carrier-added (e.g., 
reactor-produced Re-186), the latter requiring more perrhenate for 
equivalent activity, and therefore more stannous ion reducing agent, 
although not necessarily more protein. This is an aspect not treated in 
the prior art. 
Generally, a protein, preferably an antibody or antibody fragment, 
containing a plurality of adjacent/proximal sulfhydryl groups, will be 
used in concentrations reflecting the preferred therapy application of 
radioisotopes of rhenium. The types of protein that can be labeled and the 
definitions of antibodies and antibody fragment disclosed for technetium 
labeling are also valid for rhenium radiolabeling. 
Labeling with substantially carrier-free Re-188-NaReO.sub.4, the form which 
would normally be produced from a generator, is advantageously effected 
with the sulfhydryl-containing protein, e.g., antibody or fragment, at a 
protein concentration of about 1-20 mg/ml, preferably 10-20 mg/ml, in 
substantially the same solutions and under substantially the same 
conditions as pertechnetate. The amount of stannous ion used is generally 
about 100-10,000 .mu.g/ml, preferably about 500-5,000 .mu.g/ml, and 
preferably in proportion to the amount of protein. Using the foregoing 
amounts of protein and stannous ion, it is advantageous to use about 
10-500 mCi , preferably about 50-250 mCi of substantially carrier-free 
Re-188-perrhenate, preferably again in proportion to the amount of 
protein. The reaction time is advantageously about 1 min to 4 hr, 
preferably about 15 min to 2 hr. Surprisingly and unexpectedly, the 
reagent ratios and reactions times that were optimal for pertechnetate 
labeling were not effective for perrhenate, and vice-versa. 
Labeling with carrier-added Re-186-NaReO.sub.4, the form which would 
normally be produced from a reactor and which generally contains about a 
100- to 1,000-fold excess of Re-185 as carrier, is advantageously effected 
with the sulfhydryl-containing protein, e.g., antibody or fragment, at a 
protein concentration of about 1-20 mg/ml, preferably 10-20 mg/ml, in 
substantially the same solutions and under substantially the same 
conditions as for Re-188-perrhenate. However, because of the large amount 
of carrier, the amount of stannous ion used is generally about 1-1,000 
mg/ml, preferably about 5-500 mg/ml, and preferably again in proportion to 
the amount of protein. Approximately the same activity of rhenium 
radioisotope and about the same reactions times are used for this isotope. 
A short column procedure will normally suffice to remove any unbound 
rhenium and after this time it is ready for immediate injection, if done 
in a properly sterilized, pyrogen-free vial. Again, no blocking of free 
sulfhydryl groups after rhenium labeling is necessary for stabilization. 
In a surprising and unexpected development, it has now been shown that 
proteins containing at least one disulfide group, e.g., intact antibodies 
(without prior reduction) can be simply and directly radiolabeled with 
rhenium using a larger amount of stannous ion to concommitantly reduce and 
bind together the antibody and the rhenium species. The "pretinning" 
procedures described elsewhere and earlier technetium-IgG labeling 
procedures gave poor results. Long and/or severe reactions are 
incompatible with the successful generation of an IgG-rhenium injectible. 
In general, the concentration of unreduced protein, e.g., antibody, the 
reaction times, perrhenate activities and other conditions will be 
substantially the same as for Re-186 or Re-188 labeling of 
sulfhydryl-containing proteins, except that a larger amount of stannous 
ion is used. When the radioisotope in the radioperrhenate is substantially 
carrier-free Re-188, the concentration of antibody or antibody fragment in 
the solution is advantageously about 1-20 mg/ml, preferably about 10-20 
mg/ml, and the amount of stannous ion is about 500-10,000 .mu.g/ml, 
preferably about 500-5,000 .mu.g/ml. When the radioisotope in the 
radioperrhenate is carrier-added Re-186, at the same concentration of 
antibody or antibody fragment, the amount of stannous ion is about 5-1,000 
mg/ml, preferably about 50-500 mg/ml. 
In fact, unmodified, unreduced IgG has been taken, placed in a vial with a 
stannous reductant and successfully labeled with perrhenate in as little 
as 45 minutes at room temperature. No pretinning is used, but perrhenate 
is added directly after the antibody and tin are mixed. The key is to have 
sufficient tin to effect a rapid reduction. A short separatory column, 
simpler in ease of operation than a typical iodine-131 label purification, 
gives a pure rhenium-IgG conjugate ready for injection. 
Exposure of IgG to the conditions used does not impair its immunoreactivity 
as measured on an affinity column of bound antigen. It appears that an in 
situ reduction of protein disulfide groups by the stannous ion accompanies 
the perrhenate reduction and creates the necessary conditions for 
protein-metal complex formation. The fact that pertechnetate labels IgG 
much less favorably under the same conditions suggests this as a 
particularly novel and efficient route for obtaining rhenium antibodies 
with minimal manipulation. 
Rhenium labeling is effected in substantially the same manner as technetium 
labeling, with special care being taken to insure the absence of air or 
oxygen in the system. The rhenium-labeled proteins prepared according to 
the invention show no sign of the ready reoxidation to perrhenate seen by 
other workers, indicating that the present method is not only facile but 
also stable. Coupled with this are the facts that much less IgG 
manipulation is needed for the present method, that Re-188 is available in 
a convenient generator format (with a single generator capable of daily 
use for a period of sixty days or more) and that no problems are 
encountered with strontium contamination, making rhenium-radiolabeled IgG 
an attractive therapeutic agent. 
Rhenium antibody conjugates produced by these methods have been shown to be 
very stable, even when exposed in solution to the atmosphere for up to 5 
days at least. This long term stability is important in an 
immunoradiotherapeutic, as is retention of immunoreactivity during 
labeling procedures, which also has been demonstrated. 
It must be recognized that the present approach is quite different from 
prior art approaches, due to the simplicity, effectiveness, efficiency and 
lack of major manipulation in the process as well as the stability of the 
rhenium conjugates. It is again emphasized that the relatively higher 
amount of tin and longer reaction times are not conducive to efficient 
pertechnetate labeling but are optimal for perrhenate, whereas the low 
tin, fast labeling conditions optimal for pertechnetate do not work for 
perrhenate. In particular, for about 1 mg of antibody, and an imaging 
activity of Tc-99m, very low tin and 5 min reaction times result in 
excellent results, whereas for therapy levels of Re-186 or Re-188 label, 
more than 500 .mu.g/ml stannous ion is desirable, especially if intact 
antibody is used, and reaction times on the order of close to an hour are 
effective. 
A kit for use in radiolabeling a protein, e.g., a Fab'-SH or F(ab)'.sub.2 
fragment or an intact antibody, with Tc-99m, using generator-produced 
pertechnetate, would include, e.g., in separate containers: about 0.01-10 
mg per unit dose of an antibody or antibody fragment which specifically 
binds an antigen associated with a tumor, an infectious lesion, a 
microorganism, a myocardial infarction, a clot, atherosclerotic plaque, or 
a normal organ or tissue, and which contains a plurality of adjacent free 
sulfhydryl groups; about 0.1-50 .mu.g per unit dose of stannous ions; and 
about 2-50 mCi of Tc-99m pertechnetate per mg of antibody or antibody 
fragment. 
A kit for radiolabeling an antibody or antibody fragment with the Re-188 
radioisotope of rhenium would typically include, in separate containers: 
about 1-20 mg per unit dose of an antibody or antibody fragment which 
specifically binds an antigen associated with a tumor or an infectious 
lesion, and which contains a plurality of adjacent free sulfhydryl groups; 
about 100-10 000 .mu.g per unit dose of stannous ions; and about 10-500 
mCi of substantially carrier-free Re-188 perrhenate per mg of antibody or 
antibody fragment. 
A kit for radiolabeling an antibody or antibody fragment with the Re-186 
radioisotope of rhenium would typically include, in separate containers: 
about 1-20 mg per unit dose of an antibody or antibody fragment which 
specifically binds an antigen associated with a tumor or an infectious 
lesion, and which contains a plurality of adjacent free sulfhydryl groups; 
about 1-1,000 mg per unit dose of stannous ions; and about 10-500 mCi of 
carrier-added Re-186 perrhenate per mg of antibody or antibody fragment. 
A kit for labeling unreduced intact antibody with either rhenium isotope 
would be similar to the foregoing, except for the larger amount of 
stannous ions generally used to reduce some of the disulfide bonds in the 
antibody as well as reducing the perrhenate. The proteins in such kits are 
advantageously frozen or lyophilized, in sterile containers, and under an 
inert gas atmosphere. The kits are conveniently supplemented with sterile 
vials of buffers, saline, syringes, filters, columns and the like 
auxiliaries to facilitate preparation of injectable preparations ready for 
use by the clinician or technologist. 
Additional significantly improved reagents and conditions for a kit and 
method for "instant" Tc-99m labeling of monovalent, e.g., Fab or Fab', 
antibody fragments containing at least one and preferably a plurality of 
spatially adjacent stabilized free sulfhydryl groups, have also been 
provided. Labeling is effected substantially quantitatively at ambient 
temperature within about 5 minutes of mixing a solution of antibody 
fragment with pertechnetate, readily available from commercial generators. 
In this second method according to the invention, the stabilized Fab-SH or 
Fab'-SH fragments are next mixed with stannous ion, preferably stannous 
chloride, and with a stabilizer for the stannous ions. In the second 
method of the invention, it can be added in the form of SnCl.sub.2, 
advantageously in a solution that is also about 0.01 N in HCl in a ratio 
of about 100-150, preferably about 123 .mu.g Sn per mg of fragment. 
Advantageously, the stannous ion solution is prepared by dissolving 
SnCl.multidot.2 H.sub.2 O in 6 N HCl and diluting the resultant solution 
with sterile H.sub.2 O that has been purged with argon. 
A stabilizing agent for the stannous ion is advantageously present in the 
solution. It is known that ascorbate can improve specific loading of a 
chelator with reduced pertechnetate and minimize formation of TcO.sub.2, 
when the reducing agent is stannous ion. Other polycarboxylic acids, e.g., 
tartrate, citrate, phthalate, iminodiacetate, ethylenediaminetetraacetic 
acid (EDTA), diethylenetriaminepentaacetic acid (DTPA) and the like, can 
also be used. Although polycarboxylic acids are mentioned, by way of 
illustration, any of a variety of anionic and/or hydroxylic 
oxygen-containing species could serve this function, e.g., salicylates, 
acetylacetonates, hydroxyacids, catechols, glycols and other polyols, 
e.g., glucoheptonate, and the like. Preferred such stabilizers are 
ascorbate, citrate and tartrate, more preferably tartrate. 
While the precise role of such agents is not known, it appears that they 
chelate stannous ion and may prevent adventitious reactions and/or promote 
reduction by stabilization of stannic ions, and they may also chelate--and 
thereby stabilize--certain oxidation states of reduced pertechnetate, 
thereby serving as transchelating agents for the transfer of these 
technetium ions to the presumably more stable chelation with one or more 
thiol groups and other nearby ligands on the protein. Such agents will be 
referred to as "stabilizers" herein. The molar ratio of stabilizer to 
stannous ion is about 30:1-40:1. 
A solution of stabilizer, e.g., NaK tartrate, advantageously at a 
concentration of about 0.1 M, in buffer, preferably sodium or ammonium 
acetate at a pH of about 5.5, is prepared with sterile H.sub.2 O purged 
with argon. One volume of the SnCl.sub.2 solution is mixed with enough of 
the stabilizer solution to provide a 30-40 molar excess, relative to the 
stannous ion, and the resultant solution is sterile filtered and purged 
with argon. 
The sterile, stabilized SnCl.sub.2 solution is mixed with the sterile 
Fab'-SH or Fab-SH solution to obtain a final concentration of about 
100-150, preferably about 123 .mu.g Sn per mg of fragment. The pH is 
adjusted, if necessary to about 4.5-4.8. 
The solution of fragment and stabilized stannous ion is advantageously 
metered into sterile vials, e.g., at a unit dosage of about 1.25 mg 
fragment/vial, and the vials are either stoppered, sealed and stored at 
low temperature, preferably in liquid nitrogen, or lyophilized. In the 
latter case, the buffer is advantageously ammonium acetate and the 
solution is made about 0.09 molar with a sugar such as trehalose or 
sucrose, preferably sucrose, prior to metering into sterile vials. The 
material in the vials is then lyophilized, the vacuum is broken with an 
inert gas, preferably argon, and the vials containing the lyophilizate are 
stoppered, sealed and stored, optionally in the freezer. The 
lyophilization conditions are conventional and well known to the ordinary 
skilled artisan. Both the sealed lyophilizate and the sealed liquid 
nitrogen stored solution are stable for at least 9 months and retain their 
capacity to be rapidly and quantitatively labeled with Tc-99m ions upon 
mixing with pertechnetate. 
To label a unit dose of antibody fragment, a vial of liquid nitrogen frozen 
solution is thawed to room temperature by gentle warming, or a vial of 
lyphilizate is brought to ambient temperature if necessary, and the seal 
is broken under inert gas, preferably argon. A sterile saline solution of 
a suitable imaging quantity of pertechnetate is added to the vial and the 
contents are mixed. When labeling the foregoing unit dosage quantity of 
antibody fragment, the amount of pertechnetate is generally about 1-50 
mCi/mg of antibody fragment, and the time of reaction is about 0.1-10 min. 
With the preferred concentrations of protein and stannous ions noted 
above, the amount of pertechnetate is preferably about 5-15 mCi/mg, and 
the time of reaction is preferably about 1-5 min. This is effectively an 
"instant" labeling procedure with respect to the prior art processes which 
generally required 30 minutes to several hours incubation, in some cases 
at elevated temperatures and/or with additional purification required. 
This process also routinely results in substantantially quantitative 
incorporation of the label into the antibody fragment in a form which is 
highly stable to oxidation and resistant to transchelation in saline and 
serum. When labeled with Tc-99m according to the method of the present 
invention, 100% incorporation of Tc-99m to Fab' is seen (within the limits 
of detection of the analytical monitor) together with &gt;95% retention of 
immunoreactivity. The radioantibody solutions as prepared above are ready 
for immediate injection, if done in a properly sterilized, pyrogen-free 
vial. Also, no blocking of free sulfhydryl groups after technetium binding 
is necessary for stabilization. Furthermore the immunoreactivity of the 
labeled fragment is hardly reduced after serum incubation for a day, 
showing that the conjugates are still completely viable imaging agents out 
to at least 24 hours. 
A kit for use in radiolabeling a monovalent antibody fragment, e g., an 
Fab'-SH or Fab-SH fragment, with Tc-99m, using generator-produced 
pertechnetate, in accordance with the foregoing method, would typically 
include about 0.01-10 mg, preferably about 1-2 mg, per unit dose of an 
antibody fragment which specifically binds an antigen, and which contains 
at least one but preferably a plurality of adjacent free sulfhydryl 
groups; about 100-150 .mu.g per mg of fragment of stannous ions, and a 
30-40 molar excess, relative to the stannous ions, of a stabilizer such as 
tartrate. The constituents of the kit are provided in a single, sealed 
sterile vial, in the form of a solution or a lyophilizate, and are mixed 
just prior to use with about 2-50 mCi of Tc-99m pertechnetate per mg of 
antibody or antibody fragment. Normally, the kit is used and/or provided 
in combination with one or more auxiliary reagents, buffers, filters, 
vials, columns and the like for effecting the radiolabeling steps. 
Variations and modifications of these kits will be readily apparent to the 
ordinary skilled artisan, as a function of the individual needs of the 
patient or treatment regimen, as well as of variations in the form in 
which the radioisotopes may be provided or may become available. 
It will also be apparent to one of ordinary skill that the radiolabeled 
proteins, especially antibodies and antibody fragments, prepared according 
to the method of the invention, will be suitable, and in fact particularly 
convenient and efficacious, in methods of non-invasive scintigraphic 
imaging and for radioantibody therapy of tumors and lesions. In 
particular, in a method of imaging a tumor, an infectious lesion, a 
myocardial infarction, a clot, atherosclerotic plaque, or a normal organ 
or tissue, wherein an antibody or antibody fragment which specifically 
binds to an antigen produced by or associated with said tumor, infectious 
lesion, myocardial infarction, clot, atherosclerotic plaque, or normal 
organ or tissue, and radiolabeled with a pharmaceutically inert 
radioisotope capable of external detection, is parenterally injected into 
a human patient and, after a sufficient time for the radiolabeled antibody 
or antibody fragment to localize and for non-target background to clear, 
the site or sites of accretion of the radiolabeled antibody or antibody 
fragment are detected by an external imaging camera, it will be an 
improvement to use as the radiolabeled antibody or antibody fragment a 
Tc-99m-labeled antibody or antibody fragment made according to the method 
of the present invention. Such imaging methods are well known in the art. 
Moreover, in a method of radioantibody therapy of a patient suffering from 
a tumor or an infectious lesion, wherein an antibody or antibody fragment 
which specifically binds to an antigen produced by or associated with a 
tumor or an infectious lesion, and radiolabeled with a therapeutically 
effective radioisotope, is parenterally injected into a human patient 
suffering from such tumor or infectious lesion, it will represent an 
improvement to use as the radiolabeled antibody or antibody fragment a 
rhenium radiolabeled antibody or antibody fragment made according to the 
method of the present invention, either from pre-reduced or unreduced 
antibody. 
Without further elaboration, it is believed that one skilled in the art 
can, using the preceding description, utilize the present invention to its 
fullest extent. The following preferred specific embodiments are, 
therefore, to be construed as merely illustrative, and not limitative of 
the remainder of the disclosure in any way whatsoever. In the following 
examples, all temperatures are set forth uncorrected in degrees Celsius; 
unless otherwise indicated, all parts and percentages are by weight. 
EXAMPLE 1 
Antibody Reduction 
In a typical run, a solution of 5 mg of purified monoclonal anti-CEA IgG 
(antibody which specifically binds to carcinoembryonic antigen, a marker 
associated with colorectal cancer) in 1 ml of phosphate-buffered saline 
(PBS), at a pH adjusted to 6.2-6.6, is made 30-50 millimolar in 
2-mercaptoethanol. After standing at room temperature for 30-40 minutes, 
the sample is purified on an acrylamide column with deoxygenated acetate 
as buffer. The reduced IgG solution is concentrated to 1-2 mg/ml on 
Centricon and is analyzed for SH groups per IgG by the Ellman reaction. It 
is stored sterile filtered at 4.degree. C. for convenience or frozen for 
greater stability of the SH groups. 
EXAMPLE 2 
Tc-99m Radiolabeling 
In a typical run, a solution containing 125 nanograms of tin(II) is mixed 
with a solution containing 3.6.times.10.sup.-10 moles of monoclonal 
anti-CEA with free sulfhydryl groups, prepared according to Example 1 and 
having 1.2 SH per IgG by the Ellman reaction, giving a 2.9:1 ratio of 
tin(II) to IgG. Addition of 2 mCi of technetium-99m as pertechnetate in 
saline, followed by incubation for 5 minutes at room temperature, gives a 
100% incorporation of technetium into the protein as measured by HPLC and 
less than 1% pertechnetate remaining by ITLC in two different elution 
buffers. Immunoreactivity is &gt;98% when measured on a CEA column. 
EXAMPLE 3 
Re-186 Radiolabeling 
In a typical run, a solution containing 880 micrograms of tin(II) is mixed 
with a solution containing 1 mg of monoclonal anti-CEA intact antibody. 
Addition of 18.9.times.10.sup.-11 mol rhenium-186, with an activity of 9 
million cpm, as perrhenate in saline, followed by incubation for 1 hour at 
room temperature, gives a 75% incorporation of rhenium into the protein. 
The labeled antibody is stable to air oxidation and its immunoreactivity 
is high when measured on a CEA column. 
EXAMPLE 4 
Re-188 Labeling 
In a typical run, 1 mg of anti-CEA Fab'-SH (SH generated by reduction of 
F(ab').sub.2 with cysteine, mercaptoethanol or dithiothreitol), is placed 
in an argon atmosphere together with 1 ml of 100 mM tartrate +50 mM 
acetate buffer and an Sn(II) species containing approximately 123 ug of 
Tin(II). Then, 20-2000 .mu.l of a perrhenate solution depending on 
concentration, approximately equivalent to up to 1.times.10.sup.-9 mol 
rhenium is added. 
After mixing, the reaction is effected in 1 to 6 hours with rhenium 
incorporations of 60 to 100% seen. The labeled protein is immediately 
applied to an acrylamide column and eluted with either acetate buffer or 
saline. The labeled protein elutes with the void volume (approx. 5 ml) and 
is shown to be 100% protein-bound rhenium by ITLC in saline. The 
conjugates can be equally well purified by a quick run through a 
mini-column using a syringe barrel, again to give a 100% protein bound 
label. 
The label is put through filters down as low as 0.05 micron pore size to 
show lack of aggregate and in all column procedures and filtrations 
essentially 100% recovery of radioactivity is obtained. Chromatography on 
HPLC and ITLC shows the radioactivity eluting/migrating with the protein 
fraction. 
EXAMPLE 5 
Preparation of Tc-99m-anti-CEA-Fab' 
A. Labeling Kit 
The following solutions are prepared. 
(I) A solution of 0.075 M SnCl.sub.2 is prepared by dissolving 3350 mg 
SnClK2 H.sub.2 O in 1 ml of 6 N HCl and diluting the resultant solution 
with sterile H.sub.2 O that has been purged with argon. 
(II) A solution of 0.1 M NaK tartrat in 0.05 M NaAc, at pH 5.5, is prepared 
with sterile H.sub.2 O purged with argon. 
(III) One volume of solution I is mixed with 26 volumes of solution II, and 
the resultant solution is sterile filtered and purged with argon. 
A solution of anti-CEA-Fab'-SH, prepared from a murine monoclonal IgG.sub.1 
antibody that specifically binds to carcinoembryonic antigen (CEA) by 
pepsin cleavange to an F(ab').sub.2 fragment, is reduced to Fab'-SH with 
20 mM cysteine; excess cysteine is removed by gel filtration, and the 
Fab'-SH is stabilized (2 mg/ml) at pH 4.5 in 0.05 M NaOAc buffer which is 
0.15 M in saline; and the resultant solution is sterile filtered and 
purged with argon. 
(V) Mix solution IV with enough of solution III to obtain a final 
concentration of 123 .mu.g Sn per mg of Fab'-SH, and adjust the pH to 
4.5-4.8. 
Solution V is filled, under argon, into sterile vials (1.25 mg Fab'-SH per 
vial), stoppered and crimp-seal, and the vials are stored in liquid 
nitrogen. 
Alternatively, NH.sub.4 OAC is used instead of NaOAc, solution V is made 
0.09 M with sucrose, the resultant solution is filled, under argon, into 
sterile vials (1.25 mg Fab'-SH per vial) and lyophilized. The vacuum is 
broken with argon, and the vials containing the lyophilizate are stoppered 
and crimp-sealed. 
B. Labeled Fragment 
A vial of liquid nitrogen stored fragment is gently warmed, or a vial of 
lyophilizate prepared according to part A above is selected. A sterile 
solution of 10 mCi of sodium pertechnetate in sterile saline from a 
generator is injected into the vial of Fab'-SH and stabilized stannous 
ions and mixed by gentle agitation. Labeling is quantitative in five 
minutes, and the resultant solution of Tc-99m-labeled fragment is ready 
for immediate injection into a patient. 
EXAMPLE 6 
Tumor Imaging 
A sterile solution of a unit dose of Tc-99m-labeled anti-CEA-Fab' prepared 
(with liquid nitrogen stored Fab'-SH solution) according to Example 5 is 
infused intravenously into a patient with a progressively rising CEA 
titer, the patient having undergone "curative" surgery for a colon 
carcinoma three years earlier. Scintigraphic imaging 2 hr postinjection 
demonstrates antibody fragment localization in the pelvis at the site of 
removal of the primary tumor. Subsequent surgery confirms the presence of 
a 1.0.times.0.5 cm carcinoma that is successfully removed. 
EXAMPLE 7 
Tumor Imaging 
A sterile solution of a unit dose of Tc-99m-labeled anti-CEA-Fab' prepared 
(from lyophilizate) according to Example 5 is infused intravenously into a 
patient with a 3.times.2 cm rectal polyp that has been proven by biopsy to 
be malignant. Imaging 2 hr postinjection demonstrates localized antibody 
fragment in the primary tumor, the right lobe of the liver and in the 
lower lobe of the left lung. Needle biopsy confirms the presence of tumor 
in both the liver and the lung. The original plan to perform surgery and 
adjuvant radiation therapy is abandoned and palliative chemotherapy is 
instituted. 
The foregoing examples are merely illustrative and numerous variations and 
modifications can be effected by one of ordinary skill in the art to adapt 
the method, kit and uses thereof according to the invention to various 
usages and conditions without departing from the scope and spirit of the 
invention. 
The broad scope of the invention is defined by the appended claims, and by 
the penumbra of equivalents thereof.