In vivo lymphocyte tagging

Methods and reagents for the in vivo tagging of leukocytes, and in particular lymphocytes with a leukostimulatory agent and a linked medically useful metal ion, including a radioisotope, and subsequent detection of leukocyte or lymphocyte trafficking and sites of concentrated leukocytes or lymp PAC LICENSE RIGHTS The U.S. Government has a paid-up license in this invention and the right in limited circumstances to require the patent owner to license others on reasonable terms as provided for by the terms of Small Business Innovative Research Grant No. 1 R43 AR41124 awarded by the National Institute of Arthritis and Musculoskeletal and Skin Diseases, Department of Health and Human Services.

BACKGROUND OF THE INVENTION 
1. Field of the Invention (Technical Field): 
The present invention relates to the in vivo tagging of leukocytes, and in 
particular lymphocytes, with radioactive tracers and other diagnostically 
useful metal ions, and subsequent detection of lymphocyte trafficking and 
sites of concentrated lymphocytes within the mammal by radiodetection or 
other means. 
2. Description of the Related Art, Including Information Disclosed under 37 
C.F.R. Sections 1.97-1.99 (Background Art): 
This application covers generally the development of a pan-T-lymphocyte 
tracer that can be used both to study trafficking of stimulated 
lymphocytes and as a diagnostic radiopharmaceutical or magnetic resonance 
imaging diagnostic pharmaceutical, for use in chronic infections such as 
osteomyelitis and granulomatous diseases and for other conditions. One of 
the disadvantages of using only monoclonal antibody tracers to tag 
circulating lymphocytes is that they are generally species specific, and a 
monoclonal antibody that recognizes a specific subset of human lymphocytes 
can be used only for studies in humans. Thus, there is a need for specific 
tracers that can be used in both laboratory animals and man, so that 
experimental studies, the results of which will be extrapolated to humans, 
can be conducted. 
One class of pan-T-lymphocyte tracers disclosed herein are leukostimulatory 
lectins, and particularly the plant-derived leclin. phytohemagglutinin-L4 
(PHA-L4). This isolectin binds the CD3 receptor on T-lymphocytes of both 
laboratory animals and humans. Wimer, B. M., "The ideal biological 
response modifier," Mol Biother 1:311-317, 1989. It also stimulates the 
lymphocytes to differentiate and divide (Wimer, B. M., "Characteristics of 
PHA-L4, the mitogenic isolectin of phytohemagglutinin, as an ideal 
biologic response modifier," Mol Biother 2:4-17, 1990); thus, the labeled 
molecule provides a tracer that can be used to track stimulated 
lymphocytes. 
Although the use of PHA as a drug has been studied for many years (See 
generally, Wimer, B. M., "Therapeutic activities of PHA-L4, the mitogenic 
isolectin of phytohemagglutinin," Mol Biother 2:74-90, 1990; and, Wimer, 
B. M., "Potential therapeutic applications of PHA-L4, the mitogenic 
isolectin of phytohemagglutinin," Mol Biother 2:196-200, 1990), it has not 
found a permanent place in the routine practice of medicine. 
Native PHA consists of four subunits and has both leuko-and 
erythro-agglutinating properties, as well as leukostimulatory properties. 
The L4 isolectin of PHA carries only the leukoagglutinating and 
stimulating species, and has been found to bind the CD3 receptor on 
T-lymphocytes. Greaves, M. F., Bauminger, S., Janossy, G., "Lymphocyte 
activation. III. Binding sites for phytomitogens on lymphocyte 
subpopulations," Clin Exp ImmunoI 10:537-554, 1972. 
PHA was originally isolated as an aqueous extract of the beans of the genus 
Pgaseolus, especially the red kidney bean, Phaseolus vulgaris. At least 
two active ingredients have been identified in the extract, a mucoprotein 
and a glycoprotein. The L-4 isolectin appears to be one of five isolectins 
that comprise the PHA glycoprotein. PHA may be prepared by a number of 
means, and is commercially available from E-Y Laboratories in a variety of 
forms, including the glycoprotein tetramer and the purified L-4 isolectin 
of PHA. 
PHA-L4 may also be prepared using recombinant DNA technology. Hoffman and 
Donaldson have reported the methodology for synthesis of 
non-erythroagglutinating PHA-L4 in Escherichia coli. Hoffman, L. M., and 
Donaldson, D. D., "Synthesis of mitogenic phytohemagglutinin-L in 
Escherichia coli," Biotechnology 5:157-160, 1987; Hoffman, L. M., U.S. 
Pat. No. 4,870,015, Method and Composition for Producing Lectin in 
Microorganisms. 
One advantage of using labeled PHA-L4 or other lectin-based 
leukostimulatory agents in place of monoclonal antibody tracers is the 
possibility of using the leukostimulatory agent, such as PHA-L4 isolectin, 
in several species. Generally speaking, monoclonal antibodies against the 
human CD3 receptor do not bind to murine CD3 receptors, and this lack of a 
lymphocyte tracer applicable to the laboratory mouse has limited many 
basic studies of adoptive immunotherapy. 
Rosenberg (Rosenberg, S., "Lymphokine-activated killer cells: A new 
approach to immunotherapy of cancer," JNCI 75:595-603, 1985) notes that 
immunologically active cells tend to be larger than normal resting cells. 
They are also different in other aspects, such as their level of oxidative 
metabolism, degranulation, and adherence. el-Hag, A., Clark, R. A., 
"Immunosuppression by activated human neutrophils. Dependence on the 
myeloperoxidase system," J Immunol 139:2406-2413, 1987. Thus, it appears 
that cells that are stimulated and labeled with radiolabeled PHA-L4 will 
traffic differently than the general population of lymphocytes, which can 
be labeled by methods such as .sup.111 In oxine. Thakur, M. L., Coleman, 
R. E., Welch, M. J., "Indium-111-labeled leukocytes for the localization 
of abscesses: preparation, analysis, tissue distribution, and comparison 
with gallium-67 citrate in dogs," J Lab Clin Med 89:217-228, 1977; and, 
Thakur, M. L., Lavender, J. P., Arnot, R. N., et al, "Indium-111-labeled 
autologous leukocytes in man," J Nucl Med 18:1014-1021, 1977. 
In vivo imaging with .sup.111 In-labeled lymphocytes in a study of patients 
with Hodgkin's disease was demonstrated by Lavender et al in 1977. 
Lavender, J. P., Goldman, J. M., Arnot, R. N., Thakur, M. L., "Kinetics of 
indium-111 labeled lymphocytes in normal subjects and patients with 
Hodgkin's disease, "Brit Med J 2:797-799, 1977. Such studies have been 
limited by the extreme radiosensitivity of lymphocytes labeled internally 
with .sup.111 In. A more recent approach has been to use radiolabeled 
antibodies that bind to cell surface antigens to label the cells in such a 
way that the radionuclide is distanced from the cell nucleus. Thakur, M. 
L., U.S. Pat. No. 4,917,878, Novel Use of a RadiolabeIIed Antibody Against 
Stage Specific Embryonic Antigen for the Detection of Occult Abscesses in 
Mammals; Houston, L. L., Nowinski, R. C., Bernstein, I. D., "Specific in 
vivo localization of monoclonal antibodies directed against the Thy 1.1 
antigen," J Immunol 125:837-843, 1980; and, Loutfi, I., Chisholm, P. M., 
Bevan, D., Lavender, J. P. "In vivo imaging of rat lymphocytes with an 
indium 111-labeled anti-T cell monoclonal antibody: a comparison with 
indium-111-labeled lymphocytes," Eur J Nucl Med 16:69-76, 1990. 
Labeled lymphocytes are useful for studying lymphocyte trafficking and for 
diagnostic imaging of chronic infections and granulomas, and some tumors. 
Wagstaff, J., Gibson, C., Thatcher, N., et al, "A method for following 
human lymphocyte traffic using Indium-111 oxine labeling," Clin Exp 
Immunol 43:443-449, 1981; and, Thakur, M. L., "A look at radiolabeled 
blood cells," Nucl Med Biol 13:147-158, 1986. 
The use of antibodies for in vivo tagging of leukocytes is known in the 
art. Goodwin, D. A., and Meares, C. F., U.S. Pat. No. 4,634,586, Reagent 
and Method for Radioimaging Leukocytes, teach a method in which leukocytes 
are radioimmunoimaged by injecting patients with an immunoreactive 
nonleukocidal conjugate of an anti-leukocyte and a gamma-emitting 
radioactive metal chelate, waiting for the conjugate to localize on the 
leukocytes, injecting the patient with an antibody to the conjugate to 
clear the blood of background nonlocalized conjugate and visualizing the 
leukocytes by scintillation scanning. The method can also be used without 
the step of injecting the second antibody to clear background nonlocalized 
antibody. Thakur, M. L., U.S. Pat. No. 4,917,878, Novel Use of a 
Radiolabelled Antibody Against Stage Specific Embryonic Antigen for the 
Detection of Occult Abscesses in Mammals, teaches a method whereby 
antibodies against a particular antigen found on human granulocytes, stage 
specific embryonic antigen-1, are radiolabeled using a bifunctional 
chelating agent, and the resulting radiolabeled antibody reagent injected 
under conditions which allow the reagent to accumulate at sites of occult 
abscess. 
The use of radioisotopes to label biologically derived substances is well 
known. These compositions can be used in assays, can be administered to 
the human body to visualize or monitor functioning of various parts of the 
body, and can be used for therapy. A variety of radioisotopes, including 
isotopes of iodine, technetium, indium, gallium, yttrium and rhenium have 
been used. 
Different methods can be used to radiolabel biological substances with 
radioisotopes. For iodine, a variety of iodination methods, such as 
chloramine-T, iodine monochloride, enzymatic iodination, electrolytic 
procedures and conjugation labeling, are well recognized. (Pettit, W. A., 
et al, "Iodination and acceptance testing of antibodies," Tumor Imaging: 
The Radioimmunochemical Detection of Cancer, S. W. Burchiel and B. A. 
Rhodes, Eds., Masson Publishing USA Inc., New York, 1982, pp 99-109.) 
Bifunctional chelate methods, such as DTPA conjugation, can be used to 
label antibodies with .sup.99m Tc, .sup.111 In, .sup.67 Ga, .sup.68 Ga or 
similar radionuclides. (Wensel, T. G. and Meares, C. F., "`Bifunctional` 
Chelating Agents for Binding Metal Ions to Proteins," Radioimmunoimaging 
and Radioimmunotherapy, S W Burchiel and B A Rhodes, eds., Elsevier 
Publishing Co., New York, 1983, pp 185-196.) The bifunctional chelate 
method was introduced by Krejcarek, G. E. and Tucker, K. L. (Biophys Res 
Comm 77:581-585, 1977) and has been widely employed in many variations 
using a broad variety of bifunctional chelating agents. 
U.S. Pat. No. 4,479,930 to Hnatowich, D. J., discloses methods of 
radiolabeling using a dicyclic dianhydride of compounds such as 
ethylenediaminetetraacetic acid or diethylenetriaminepentaacetic acid. The 
patent also discloses chemical compositions containing the chelating 
agents and proteins or polypeptides. U.S. Pat. No. 4,668,503 to Hnatowich, 
D. J., discloses a process for labeling amines with .sup.99m Tc in the 
presence of a stannous reducing agent. U.S. Pat. No. 4,622,420 to Meares, 
C. F., et al, discloses chelating agents which are analogs of 
ethylenediaminetetraacetic acid, ethylenediaminatriacetic acid or 
ethylenediaminepentaacetic acid which are useful in attaching radiolabels 
to biological molecules. Numerous other methods of labeling proteins and 
like substances which include lysine-containing amino acid groups, 
including those disclosed by Haber, E., and Khaw, B. A., U.S. Pat. No. 
4,421,735; and by Fritzberg, A. R., and Kasina, S., U.S. Pat. No. 
4,670,545, and by Baidoo, K. E., et al, ".sup.99m Tc Labeling of Proteins: 
Initial Evaluation of Novel Diaminedithiol Bifunctional Chelating 
Agent,"Cancer Res (Supp) 50:799s-803s, 1990, are well known in the art. A 
review article by Fritzberg et al discusses the general bifunctional 
chelate methods which may be used (Fritzberg, A. R., Berninger, R. W., 
Hadley, S. W., and Wester, D. W., "Approaches to radiolabeling of 
antibodies for diagnosis and therapy of cancer," Pharm Res 5:325-334, 
1988). 
Another general approach is direct labeling, which works with antibodies 
and other proteins containing accessible disulfide bonds or monosulfides. 
Although several direct methods have been reported, the first direct 
method capable of providing a sufficiently strong bond between the protein 
and the .sup.99m Tc for in vivo applications was the direct or pretinning 
method described in U.S. Pat. No. 4,424,200, entitled Method for 
Radiolabeling Proteins with Technetium99m, to Crockford, D. R., and 
Rhodes, B. A. In this method, a single reduction compound, consisting of 
stannous [Sn(II)]chloride and other salts which serves both to reduce the 
protein, thereby exposing the disulfide bonds, and to reduce the sodium 
pertechnetate, is used. With this method, many proteins can be 
successfully radiolabeled with .sup.99m Tc In U.S. Pat. No. 5,078,985, 
entitled Radiolabeling Antibodies and Other Proteins with Technetium or 
Rhenium by Egulated Reduction, to Rhodes, B. A., a method is provided in 
which any of a variety of reducing agents can be used to reduce disulfide 
bonds, the reducing agent is then removed, a source of Sn(II) added, and 
the preparation then labeled. 
Other methods for direct labeling have been reported on (Schwarz, A., and 
Steinstruaber, A., "A novel approach to Tc-99m-labeled monoclonal 
antibodies," J Nucl Med 28:721, 1987; Pak, K. Y., et al, "A rapid and 
efficient method for labeling IgG antibodies with Tc-99m and comparison to 
Tc-99m Fab'". J Nucl Med 30:793, 1989; Granowska, M., et al, "A 
Tc-99m-labeled monoclonal antibody, PRIA3, for radioimmunoscintigraphy," J 
Nucl Med 30:748, 1989; Reno, J. W., U.S. Pat. No. 4,877,868, Radionuclide 
Antibody Coupling). In the equivalent methods disulfide reducing agents 
other than stannous salts were used. Pak et al used dithiothreitol to 
reduce the disulfide bonds of the antibody; Swartz and Steinsbruaber, and 
Granowska et al used 2-mercaptoethanol; Reno used dithiothreitol (DTT) to 
reduce the disulfide groups of the protein, then protected the reactive 
sulfides with Zn (II) or other sulfhydryl group derivatizing reagents. 
Also some of these investigators (Swartz and Steinsbruaber, and Granowska 
et al) reduced the .sup.99m Tc prior to adding it to the reduced antibody. 
The review by Rhodes (Rhodes, B. A., "Direct Labeling of Proteins with 
.sup.99m Tc," Nucl Med Biol 18:667-676, 1991) covers direct labeling 
methods in detail, while the reviews of Hnatowich generally cover 
radiolabeling methods (Hnatowich, D. J., "Antibody radiolabeling, problems 
and promises," Nucl Med Biol 17:49-55 (1990); and, Hnatowich, D. J., 
"Recent developments in the radiolabeling of antibodies with iodine, 
indium, and technetium," Semin Nucl Med 20:80-91, 1991). 
SUMMARY OF THE INVENTION (DISCLOSURE OF THE INVENTION) 
In accordance with the present invention, a method is provided for 
detecting concentrations of leukocytes in a mammalian patient, in which 
the patient is administered an effective amount of a reagent comprising a 
leukostimulatory agent and a linked medically useful metal ion under 
conditions which allow the reagent to bind to leukocytes, and 
concentrations of leukocytes by are detected by metal ion detection means. 
This method may be employed to locate sites of concentration of leukocytes 
which are associated with abscesses, inflammations, lesions or tumors. 
In one embodiment, the leukostimulatory agent is a lectin. Lectins which 
can be used include phytohemagglutinin, concanavalin A and pokeweed 
mitogen. In the case of phytohemagglutinin, the L4 isolectin of 
phytohemagglutinin may be used. It is also possible to use 
leukostimulatory agents which are antibody, which may be in the form of 
either monoclonal antibodies or monoclonal antibody fragments. 
A variety of types of leukocytes may be detected; one class of leukocytes 
useful in practicing this invention are lymphocytes, and particularly T 
lymphocytes. The leukostimulatory agent may bind to a CD3 receptor found 
on T lymphocytes. 
A variety of imaging methods may be use to detect concentrations of 
leukocytes by metal ion detection means. These include gamma scintigraphy, 
specific photon emission computerized tomography, positron emission 
topography and magnetic resonance imaging. Similarly, a of medically 
useful metal ions may be used; these include technetium, gallium, 
ruthenium, iodine, yttrium, lead and copper. 
The leukostimulatory agent can include a chelating agent, which may be a 
bifunctional chelating agent. Representative bifunctional chelating agents 
include cyclic anhydride of diethylenetriaminepentaacetic acid and 
diaminedithiol. It is also possible to employ a bifunctional chelating 
agent which contains one or more disulfide bonds. In the event that a 
bifunctional chelating agent with one or more disulfide bonds is used, it 
can be labeled by incubating the leukostimulatory agent containing 
disulfide bonds with a first reducing agent, the period of incubation 
being sufficient to reduce available disulfide bonds to thiolate groups 
while preventing excessive fragmentation of the protein; substantially 
removing the first reducing agent from the thiolate-containing 
leukostimulatory agent; adding a source of Sn (II) agent to the 
thiolate-containing leukostimulatory agent in a sufficient amount to form 
Sn (II)-containing and sulfur-containing complexes; and labeling the Sn 
(II)-containing and sulfur-containing complexes by adding the medically 
useful metal ion, whereby the metal ion displaces the Sn (II) agent and 
the metal ion and thiolate-containing protein form metal ion-containing 
and sulfur-containing complexes. 
In the event that a leukostimulatory agent which contains disulfide bonds 
is used, such as an antibody, or a specific monoclonal antibody or 
monoclonal antibody fragments, a different method may be used for 
detecting concentrations of leukocytes in a patient. In this method, the 
leukostimulatory agent containing disulfide bonds is incubated with a 
first reducing agent, the period of incubation being sufficient to reduce 
available disulfide bonds to thiolate groups while preventing excessive 
fragmentation of the leukostimulatory agent; the first reducing agent is 
substantially removed from the thiolate-containing leukostimulatory agent; 
a source of Sn (II) agent is added to the thiolate-containing 
leukostimulatory agent in a sufficient amount to form Sn (II)-containing 
and sulfur-containing complexes; the Sn (II)-containing and 
sulfur-containing complexes is labeled by adding a medically useful metal 
ion, whereby the medically useful metal ion displaces the Sn (II) agent 
and the metal ion and thiolate-containing leukostimulatory agent form 
metal ion-containing and sulfur-containing complexes; the patient is 
administered an effective amount of the metal ion-containing and 
sulfur-containing leukostimulatory agent under conditions which allow the 
metal ion-containing and sulfur-containing leukostimulatory agent to bind 
to leukocytes; and concentrations of leukocytes are detected by metal ion 
detection means. The metal ion detection means used for imaging may 
include gamma scintigraphy, specific photon emission computerized 
tomography, positron emission tomography and magnetic resonance imaging. 
The medically useful metal ion may include isotopes of indium, technetium, 
gallium, ruthenium, iodine, yttrium, lead and copper. This preparation may 
also be used to determine concentrations of leukocytes which are 
associated with abscesses, inflammations, lesions or tumors. 
Accordingly, it is an object of the present invention to provide one-step 
labeling for the rapid production of Tc-99m-PHA-L4 for the in vivo tagging 
and activation of T-cells so that location and movement within the body 
can be determined as a function of time. This radiopharmaceutical will 
thus be used to diagnose diseases in which the location and trafficking of 
T-cells is altered such as occurs in some chronic inflammations. 
It is a further object of the present invention to provide a means whereby 
diseases, including abscesses, inflammation, lesions or tumors, can be 
diagnosed. 
It is a further object of the present invention to provide a quick and 
reproducible method for the in vivo tagging of T-cells in both humans and 
in other animals. 
Another object of the present invention to provide a method for the 
combined tagging of T-cells with a radioisotope and to simultaneously 
cause T-cells to become activated. 
Another object of the present invention to provide a method for the 
combined tagging of T-cells with a paramagnetic metal ion and to 
simultaneously cause T-cells to become activated. 
Another object of the present invention is to provide labeling kits which 
can be used for radiolabeling with radioisotopes of Tc, Re, Cu, Au, Pb, 
As, Hg, Ag and other radiometals which are gamma or positron emitters and 
may be useful for diagnostic imaging. 
Other objects, advantages and novel features, and further scope of 
applicability of the present invention will be set forth in part in the 
detailed description to follow and in part will become apparent to those 
skilled in the art upon examination of the following, or may be learned by 
practice of the invention. The objects and advantages of the invention may 
be realized and attained by means of the instrumentalities and 
combinations particularly pointed out in the appended claims.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS OF THE INVENTION (BEST 
MODES FOR CARRYING OUT THE INVENTION) 
Using the methods of this invention, a leukostimulatory agent which binds 
to leukocytes may be radiolabeled and used to diagnosis conditions in 
humans and other mammals involving infections, inflammation, tumors, 
lesions, and similar conditions, and to study the trafficking of 
stimulated lymphocytes. One preferable class of leukostimulatory agents is 
lectins, with the preferable lectin being the plant-derived lectin, 
phytohemagglutinin, and particularly the L4 isolectin of 
phytohemagglutinin (PHA-L4), which binds the CD3 receptor on 
T-lymphocytes. Other isotypes and forms of phytohemagglutinin may be 
employed, including those comprising an aqueous extract of the beans of 
the genus PhaseoIus, especially the red kidney bean, Phaseolus vulgaris. 
In addition, synthetic or genetically engineered constructs may be 
employed that functionally act like PHA-L4, or other leukostimulatory 
agents, by binding to a lymphocyte membrane antigen to form a stimulatory 
complex. The term "PHA-L4" as used throughout the specification and claims 
is intended to include all of the foregoing. Other lectins and mitogenic 
substances, derived from plants, animal tissues or micro-organisms, may be 
employed, provided they are leukostimulatory, including concanavalin A 
(Con A) and pokeweed mitogen. 
The leukostimulatory agent may also be an antibody, including whole 
antibodies and antibody fragments, of any species, and including both 
polyclonal and monoclonal antibodies made by any means, as well as 
chimeric and genetically engineered antibodies, and antibody fragments of 
all of the foregoing. This includes immunoglobulins of any class, such as 
IgG, IgM, IgA, IgD or IgE; of any species origin, including human beings; 
chimeric antibodies or hybrid antibodies with dual or multiple antigen or 
epitope specificities; fragments of all of the foregoing, including 
F(ab').sub.2, F(ab).sub.2, Fab', Fab and other fragments, including hybrid 
fragments; and further includes any immunoglobulin or any natural, 
synthetic or genetically engineered protein that functionally acts like an 
antibody by binding to a specific lymphocyte membrane antigen to form a 
stimulatory complex. The term "antibody" or "antibodies", as used 
throughout the specification and claims is intended to include all such 
antibodies and antibody fragments. 
The term "leukostimulatory" as used throughout the specification and claims 
is intended to include substances which cause leukocytes, including 
lymphocytes (B cells, T cells, T cell subsets and the like), granulocytes, 
monocytes, and the like, to become immunologically active. Most uses of 
the invention will involve lymphocytes, and in particular T cell subsets. 
The term "patient" is intended to denote a mammalian individual. The 
primary applications of the invention involve human patients, but the 
invention may be applied to laboratory, farm, zoo, wildlife, pet or sport 
animals. 
It is also possible to synthesize a peptide which binds to a specific 
lymphocyte membrane antigen to form a stimulatory complex, and which 
constitutes a leukostimulatory agent. The peptide can be synthesized from 
amino acid building blocks, and contain a biological function domain and 
metal binding domain, with the metal binding domain including at least one 
cysteine amino acid. Regardless of the peptide used, if it does not 
contain a metal binding domain including at least one cysteine amino acid, 
it can be chemically modified by the introduction of disulfide bonds. 
In Rhodes B A, U.S. Pat. No. 5,078,985, Radiolabeling Antibodies and Other 
Proteins with Technetium or Rhenium by Regulated Reduction, a process is 
taught in which disulfide bonds are first partially reduced with stannous 
salts or other disulfide reducing agents, the resulting combination is 
purified, and a specified amount of radionuclide reducing agent is added. 
In Rhodes, B. A., U.S. Pat. Application Ser. No. 07/565,275, filed Aug, 8, 
1990, entitled Direct Radiolabeling of Antibodies and Other Proteins with 
Technetium or Rhenium, a method, product and kit is provided, wherein 
proteins containing one or more disulfide bonds are radiolabeled with 
radionuclides for use in diagnosis and treatment of a variety of 
pathologic conditions. Radiolabeling is accomplished by partial reduction 
of the disulfide bonds of the protein using Sn (II), or using other 
reducing agents followed by the addition of Sn (II), removal of excess 
reducing agent and reduction by-products, and addition of a specified 
amount of radionuclide reducing agent, such as stannous tartrate, with the 
addition accomplished in such a manner that further reduction of the 
protein is limited. The methods and kit of the '275 application are useful 
in the present invention. The discussions therein pertaining to technetium 
and rhenium are also appropriate for the other radiometals and metal ionic 
forms described herein. Accordingly, the teachings of this application are 
incorporated herein by reference. 
In Rhodes, B. A. and Zamora, P. O., U.S. Pat. Application Ser. No. 
07/816,477, entitled Direct Labeling of Antibodies and Other Proteins with 
Metal Ions, a method is taught in which a protein substrate containing 
monosulfides or disulfide bonds is labeled with a medically useful metal 
ion by the following method: 
a) incubating the protein with a reducing agent to reduce some or all of 
the disulfide bonds to thiolate groups, or to maintain monosulfides as 
thiolate groups; 
b) removing excess reducing agent from the protein substrate containing 
thiolate groups; 
c) adding a source of Sn (II) agent to the thiolate-containing protein 
preparation in an amount sufficient to form Sn (II)-containing and 
sulfur-containing complexes; and, 
d) adding a medically useful metal ion whereby the metal ion displaces the 
Sn (II) in the Sn (II)-containing and sulfur-containing complexes and the 
metal ion and thiolate-containing protein form metal ion-containing and 
sulfur-containing complexes. 
It is possible to chemically modify the protein by the introduction of 
disulfide bonds. A protein, even though it may not natively contain 
monosulfides or disulfide bonds, can be labeled with attached or complexed 
disulfide bonds. The medically useful metal ions includes ionic forms of 
the elements iron, cobalt, nickel, copper, zinc, arsenic, selenium, 
technetium, ruthenium, palladium, silver, cadmium, indium, antimony. 
iodine, rhenium, osmium, iridium, platinum, gold, mercury, thallium, lead, 
bismuth, polonium and astatine. Some medically useful metal ions are 
radioactive, such as radionuclidic isotopes of indium, gold, silver, 
mercury, technetium, rhenium and copper. The medically useful metal ion 
can also be paramagnetic. The product resulting from the application of 
this method can be used for gamma scintigraphy, specific photon emission 
computerized tomography, magnetic resonance imaging, positron emission 
tomography and radiotherapy. The discussions therein pertaining to 
medically useful metal ions are also appropriate for use with the 
leukostimulatory agents for leukocyte tagging described herein. 
Accordingly, the teachings of this application are incorporated herein by 
reference. 
In Rhodes, B. A., U.S. Pat. Application Ser. No. 07/816,476, entitled 
Direct Radiolabeling of Antibody Against Stage Specific Embryonic Antigen 
for Diagnostic Imaging, antibody against stage specific embryonic 
antigen-1 is radiolabeled by direct means with a radionuclide for use in 
detection of occult abscess and inflammation. Radiolabeling is 
accomplished by partial reduction of the disulfide bonds of the antibody 
using Sn(II), or using other reducing agents followed by the addition of 
Sn(II), removal of excess reducing agent and reduction by-products, and 
addition of a specified amount of radionuclide reducing agent, such as 
stannous tartrate. The antibody is specific for human granulocytes, and 
can be used to image sites of occult abscess and inflammation. This 
antibody is, however, not leukostimulatory. The discussions therein are 
also appropriate for use with the leukostimulatory agents for leukocyte 
tagging described herein. Accordingly, the teachings of this application 
are incorporated herein by reference. 
In Zamora, P. O. and Rhodes, B. A. a U.S. Pat. Application Ser. No. 
07/840,077, entitled Peptide-Metal Ion Pharmaceutical Preparation and 
Method, peptides containing a biological-function domain and a medically 
useful metal ion-binding domain are labeled with medically useful metal 
ions for use in diagnosis and treatment of a variety of pathologic 
conditions. The peptides have the amino acid sequence 
(R.sub.1)-[Cys].sub.n -(R.sub.2), 
(R.sub.1)-[Cys-(R.sub.2)-Cys].sub.n -(R.sub.3), 
(R.sub.1)-[Cys-(R.sub.2)-Pen].sub.n -(R.sub.3), 
(R.sub.1)-[His-(R.sub.2)-Cys].sub.n -(R.sub.3), 
(R.sub.1)-[His-(R.sub.2)-Pen].sub.n -(R.sub.3), 
or (R.sub.1)-[His-(R).sub.2 -His].sub.n -(R.sub.3) 
wherein [. . . ].sub.n is the medically useful metal ion-binding domain and 
.sub.n is a number between 1 and about 6, wherein the biological function 
domain is selected from at least one of the group consisting of R.sub.1, 
R.sub.2 and R.sub.3, and wherein R.sub.1, R.sub.2 and R.sub.3 each 
comprise an amino acid sequence containing from 0 to about 20 amino acids. 
The metal ion-binding domain of the peptide is a sequence of one or more 
amino acids containing sulfur, nitrogen or oxygen which is available for 
binding or can be made available for binding to metal ions. 
Sulfur-containing amino acids include primarily cysteine (Cys), cystine 
(Cys-Cys) and penicillamine (Pen), although deacylated methionine (Met) 
may also be used. Nitrogen-containing amino acids include primarily 
histidine (His), but under certain conditions lysine (Lys) and arginine 
(Arg), which have pK.sub.a values of 10.0 and 12.0, may also be employed. 
In addition, the terminal amino group of peptides may also be employed. 
Oxygen-containing amino acids include aspartic acid (Asp), glutamic acid 
(Glu) and tyrosine (Tyr), as well as the terminal carboxyl group of 
peptides. The amino acid sequences most usefully employed will include one 
or more Cys, one or more His, or a combination of Cys and His. Pen, which 
is an analogue of Cys, may be directly substituted for any given Cys. Cys 
may be present in the peptide as a disulfide in the form of cystine. The 
metal ion-binding domains may occur once or multiple times in any given 
peptide, and may occur in any combination. The metal ion-binding domain 
and the biological function-domain may overlap. The resulting product may 
be stored frozen or lyophilized, with labeling accomplished by the 
addition of the medically useful metal ions. The medically useful metal 
ion may be radioactive or paramagnetic, with diagnosis performed by gamma 
scintigraphy, specific photon emission computerized tomography, positron 
emission tomography or magnetic resonance imaging. The discussions therein 
are also appropriate for use with the leukostimulatory agents for 
leukocyte tagging described herein, and specifically, with peptides which 
are leukostimulatory. Accordingly, the teachings of this application are 
incorporated herein by reference. 
Because of the earlier, clinical and laboratory studies of PHA, its 
toxicity is known. The amounts needed for tracer studies, about 10-100 
micrograms per 60 kilogram subject, are far below the toxic levels. The 
LD.sub.50 for intravenously administered, native PHA in mice is 4-8 mg per 
kilogram (Weber, T., "Kinetics of the reaction of kidney-bean 
leukoagglutinin with human lymphocytes," Experientia 29:863-865, 1973), 
giving an estimated safety factor of more than 2000. In addition, at least 
part of the observed toxicity is due to the erythroagglutinating 
characteristic of native PHA, which is absent in the preferred form of 
PHA-L4 isolectin. Like monoclonal antibodies, PHA can act as an antigen, 
which occasionally could limit repeat studies in the same subject. 
Astoldi, G., Airo, R., Lisino, T., et al, "Antibodies to 
phytohaemagglutinin," Lancet 2:502-503, 1966; and, Byrd, W. J., Harek, K., 
Finley, W. H., et al, "Inhibition of the mitogenic factor in 
phytohaemagglutinin by an antiserum," Nature 213:622-624, 1967. 
It is hypothesized that stimulated lymphocytes traffic differently than do 
unstimulated lymphocytes. One of the differences appears to be that the 
ratio of stimulated lymphocytes to unstimulated lymphocytes is greater in 
lesions than in normal tissue. Lymphocytes that are simultaneously 
radiolabeled and stimulated with radiometal-PHA-L4 will accumulate in both 
inflammatory lesions and in tumors in greater numbers and ratios than will 
unstimulated lymphocytes. Because the tagging of the cells will occur in 
vivo, labeling the lymphocytes will be much simpler than with the most 
common prior art method, which requires isolating the lymphocyte fraction 
from a patient's own blood, labeling the cells with .sup.111 In oxine, and 
then reinjecting the labeled cells into the patient. The labeled cells 
will suffer less radiation damage than occurs as a result of labeling with 
.sup.111 In. 
Other lectins have leukostimulatory properties, and may be used. These 
lectins include Concanavalin A (Con A) and pokeweed mitogen. Greaves, M. 
F., Bauminger, S., Janossy, G., "Lymphocyte activation. III. Binding sites 
for phytomitogens on lymphocyte subpopulations," Clin Exp Immunol 
10:537-554, 1972. In addition to lectins, there are antibodies and other 
related substances which have leukostimulatory properties. Seaman, W. E. 
and Wofsy, D., "Selective manipulation of the immune response in vivo by 
monoclonal antibodies," Ann Rev Med 39:231-241, 1988. One such antibody is 
OKT-3, which binds to the CD3 receptor on human T-cells. 
In the preferred embodiment, diaminedithiol conjugated PHA-L4 is treated 
with stannous glucoheptonate such that there is approximately 50 .mu.gm of 
added tin in the form of Sn(II) per mg of the PHA-L4 conjugate. 
Lyoprotectants are added and the resulting mixture vialed so that 100 
.mu.gm of PHA-L4 is placed in each vial, the vials lyophilized and then 
backfilled with nitrogen gas and sealed. To prepare the 
radiopharmaceutical, up to 50 mCi of sodium pertechnetate in saline is 
added, causing the diaminedithiol conjugated PHA-L4 to come into solution, 
the pertechnetate to be reduced and the reduced technetium bound to the 
PHA-L4 by transchelation from the glucoheptonate. The Tc-99m-PHA-L4 can 
then be injected into patients for use with a gamma ray imaging device to 
determine the concentration and movement of T-cells within the body. 
In another embodiment, a leukostimulatory proteinaceous substrate, which 
may be a peptide or polypeptide, containing monosulfides or disulfide 
bonds is labeled with a medically useful metal ion by the following 
method: 
a) incubating the protein with a reducing agent to reduce some or all of 
the disulfide bonds to thiolate groups, or to maintain monosulfides as 
thiolate groups; 
b) removing excess reducing agent from the protein substrate containing 
thiolate groups; 
c) adding a source of Sn (II) agent to the thiolate-containing protein 
preparation in an amount sufficient to form Sn (II)-containing and 
sulfur-containing complexes; and, 
d) adding a medically useful metal ion whereby the metal ion displaces the 
Sn (II) in the Sn (II)-containing and sulfur-containing complexes and the 
metal ion and thiolate-containing protein form metal ion-containing and 
sulfur-containing complexes. 
The order of the steps may be altered, and the method will still produce 
metal ion-labeled proteins; the steps in the specification and claims are 
not limited to the order of steps presented. Specifically, it is possible, 
and in some cases advantageous, to add the Sn (II) to form Sn 
(II)-containing and sulfur-containing complexes prior to removing excess 
reducing agent from the protein substrate. In this way, oxidation of 
thiolate groups or reformation of disulfide bonds and other cross-linkages 
is immediately prevented. 
Any leukostimulatory protein, peptide, oliopeptide, glycopeptide, 
glycoprotein, amino acid sequence, or other substrate which contains one 
or more disulfide bonds or one or more monosulfides, including fragments 
of any of the foregoing or molecules formed by attaching or complexing any 
of the foregoing to another molecule, can be labeled as set forth above. 
Some proteinaceous substances, such as phytohemagglutinin and the L-4 
isolectin thereof, do not natively contain disulfide bonds. It is also 
possible to chemically modify the substance by the introduction of 
disulfide bonds. A proteinaceous substance, even though it may not 
natively contain monosulfides or disulfide bonds, with attached or 
complexed disulfide bonds can be labeled in accordance with this 
invention. Means to attach or complex disulfide bonds, and chelating 
agents and substrates containing disulfide bonds, are known to those 
skilled in the art. Disulfide bonds may be introduced into such proteins 
by chemical methods involving direct conjugation. Chemical means used to 
introduce disulfide bonds into proteins include use of homofunctional 
crosslinkers, heterofunctional crosslinkers, and monofunctional protein 
modification agents. Representative chemicals which can be used to 
introduce disulfide bonds into proteins include 
4-succinimidyloxycarbonyl-alpha-methyl-alpha-(2-pyridyldithiol)-toluene; 
N-succinimidyl 3-(2-pyridyldithio)propionate; sulfosuccinimidyl 
6-[3-(2-pyridiyldithiol) propinoamido] hexonate; 
dithiobis(succinimidylproprionate); 
3,3'-dithiobis(sulfosuccinimidylpropionate); and sulfosuccinimidyl 
2-(p-azidosalicylamido)ethyl-1,3'-dithiopropionate. 
The proteinaceous substrate of this invention is reacted with a medically 
useful metal ion. The medically useful metal ion may be radioactive and 
generate gamma rays, beta particles, or positrons which are converted into 
gamma rays upon collision with electrons. Alternatively, the medically 
useful metal ion may be paramagnetic. The medically useful metal ion may 
used in diagnostic imaging procedures including gamma scintigraphy, 
specific photon emission computerized tomography, or positron emission 
tomography. The medically useful metal ion may also be used in magnetic 
resonance imaging. 
The type of medically useful metal ion depends on the specific medical 
diagnostic application. Particularly attractive metal ions can be found in 
the group consisting of elements 26-30 (Fe, Co, Ni, Cu, Zn), 33-34 (As, 
Se), 42-50 (Mo, Tc, Ru, Rh, Pd, Ag, Cd, In, Sn), 53 (I) and 75-85 (Re, Os, 
Ir, Pt, Au, Hg, Tl, Pb, Bi, Po, At). The isotope .sup.99m Tc is 
particularly applicable for use in diagnostic imaging. 
For those proteinaceous substances which natively have disulfide bonds, or 
to which a disulfide bond has been attached, incubation of the protein 
with a reducing agent causes reduction of some or all of the disulfide 
bonds to thiolate groups, and in the case of proteins with monosulfides, 
causes the monosulfides to be maintained as thiolate groups. Numerous 
reducing agents have been described and are known to those skilled in the 
art. Particularly attractive types of reducing agents include 
2-mercaptoethanol; 1,4 dithiotheitol; 2,3 dihyroxylbutane-1,4-dithiol; 
2-aminoethanethiol HCl; 2-mercaptoethylamine; thioglycolate; cyanide; 
cysteine; reduced glutathione; Sn (II); Cu (I); and Ti (II). The reducing 
agent may be dissolved in a solute or may be attached to a solid phase. 
Reducing agents attached to a solid phase are commercially available, and 
methods for their use are known to those skilled in the art. The degree to 
which the protein requires disulfide bond reduction depends on the nature 
of the protein and its intended medical application. In any event, 
reduction is halted before excessive fragmentation of the protein or loss 
of the biological function of the protein occurs. 
In one specific embodiment, Sn (II) is used as a reducing agent at a 
concentration of 5 mM. In this embodiment the Sn (II) is dissolved in a 
buffer composed of 10 mM tartrate and 40 mM phthalate, pH 5.5, and the Sn 
(II) buffer admixed with a protein substrate at a concentration of 8.3 
mg/ml. The reduction reaction is allowed to proceed for 21 hours at room 
temperature, after which time the reaction is terminated by removing 
excess Sn (II) ions by molecular sieve chromatography. One means of 
molecular sieve chromatography employs Sephadex G-25, with the 
chromatography gel pre-equilibrated, and the protein eluted in 0.9% NaCl 
or other suitable buffer. 
Removal of the reducing agent, whether Sn (II) or some other reducing 
agent, can be accomplished by a variety of suitable means, including such 
methods as dialysis, ultrafiltration, positive-pressure membrane 
filtration, precipitation, preparative high performance liquid 
chromatography, affinity chromatography, other forms of chromatography and 
preparative isoelectric focusing. Many of the reducing agents contain 
thiols, which if present in the final labeling mixture, can complex with 
the medically useful metal ion. Such complexes can have severe and unknown 
side effects if administered in vivo. Additionally, some reducing agents 
exhibit unacceptable toxicity. Thus removal of the reducing agent both 
limits the degree of reduction to that desired, as well as providing for 
increased utility and safety of the labeled preparation by removal of 
toxic or otherwise undesirable reducing agents. 
Thiolate groups in reduced proteins are highly reactive and can interact to 
form disulfide bonds. The use of Sn (II) is believed to minimize the 
reformation of disulfide bonds. Sources of Sn (II) include stannous 
tartrate, stannous glucoheptonate, stannous gluconate, stannous 
phosphonate, stannous chloride, and stannous fluoride. The selection of 
the source of Sn (II) and its final concentration depends on the intended 
medical application of the protein, the nature of the protein, the 
relative and absolute number of thiolate groups and the metal ion to be 
used. In one embodiment stannous tartrate is used at a concentration of 
1.25 mM. The stannous tartrate is added to the protein after removal of 
the protein-reducing agent. The stannous tartrate is prepared in a buffer 
composed of 10 mM tartrate and 40 mM phthalate, pH 5.6, and is added to 
protein to yield a final concentration of 1 mg/ml protein solution. 
Sn (II) can be stabilized by use of dicarboxylic acids, such as phthalate 
and tartrate. A wide range of dicarboxylic acids, known to those skilled 
in the art, may be similarly used to stabilize the Sn (II) and/or to act 
as a buffer. If the phthalate and tartrate are in molar excess relative to 
the Sn (II), then these dicarboxylic acids also stabilize the medically 
useful metal ion in a form which can react with the protein. In one 
embodiment tartrate and phthalate are used in the Sn (II) agent at 
concentrations of 10 mM and 40 mM, respectively. 
Similarly, the Sn (II) and the medically useful metal ion may be stabilized 
by amino acids used singly or in combination with other agents. The type 
of amino acid used and the specific concentration depends on the nature of 
the protein and its intended use. In one embodiment, glycine is used at a 
concentration of 0.1-10 mM, and in another, histidine is used at a 
concentration of 0.1-10 mM. 
The protein may be stored frozen in bulk form after disulfide bond 
reduction and the removal of excess reducing agent. Alternatively, the 
protein may be stored in bulk form or in unit dose form after addition of 
the Sn (II). Similarly, the protein may be stored lyophilized during or 
after processing. For example, in one embodiment the protein is stored in 
vials after introduction of the Sn (II). Methods used in lyophilization of 
proteins are known to those skilled in the art. Either frozen or 
lyophilized preparations may be maintained for an indefinite period before 
labeling by the addition of the medically useful metal ion. 
In both the frozen and lyophilized storage forms, excipients may be added 
to the protein to minimize damage which can arise from ice-crystal 
formation or free-radical formation. The type of excipient and the 
concentration depends on the nature of the protein and the intended use. 
In one embodiment, glycine and inositol are used as excipients in 
lyophilized preparations. 
A typical lyophilized preparation made by the embodiments set forth above 
would, upon rehydration, contain 10 mM tartrate, 40 mM phthalate, 22 .mu.g 
of Sn (II), 500 .mu.g of protein, 2 mg/ml of glycine, and 2 mg/ml of 
inositol. The amounts of protein and Sn (II) used in the kits would depend 
on the medical application, varying depending on biodistribution of the 
protein, imaging modality being used, type of metal ion and related 
factors. Similarly, the amount and type of buffer components (such as 
tartrate and phthalate) and excipients (such as glycine and inositol) 
depends on the specific application. 
To label with a medically useful metal ion, a typical lyophilized 
preparation is hydrated by the addition of a solution containing 0.9% NaCl 
(U.S.P.) or water for injection (U.S.P.) and the medically useful metal 
ion. Alternatively, it is possible to hydrate the lyophilized preparation, 
and to add the metal ion in a subsequent step. If a frozen preparation is 
used, it is thawed and allowed to come to room temperature, and a solution 
containing the medically useful metal ion is then added. The nature and 
amount of the medically useful metal ion and the specific reaction 
conditions depend on the isotopic nature of the metal, and the intended 
medical application. In one embodiment, .sup.99m Tc is added in the form 
of pertechnetate ion in a solution of 0.9% NaCl. The .sup.99m Tc is 
typically incubated for up to 30 minutes to insure completion of the 
reaction with the protein, after which the radiolabeled preparation can be 
directly used in medical applications. In another embodiment, .sup.67 Cu 
is added in a solution of 10 mM tartrate and 40 mM phthalate at pH 5.6. In 
yet another embodiment, .sup.188 Re or .sup.186 Re is added to a solution 
of 10 mM tartrate and 40 mM phthalate, at pH 5.6, and containing Sn (II), 
and then heated to lower the oxidation state of Re. The resulting solution 
is then added to the lyophilized or frozen preparation. 
In the embodiment in which .sup.99m Tc is used, the Sn (II) is present in 
the protein-containing solution in sufficient excess to alter the 
oxidation state of the Tc ion such that it can bind to thiolate groups. 
Typically Tc (VII) is reduced to Tc (III), Tc (VI), and/or Tc (V). The 
preferred state of Tc to be added to protein preparations is as the 
pertechnetate ion, (TcO.sub.4). The Sn (II) then reacts with the 
pertechnetate ion resulting in a composition in which the Tc is in a lower 
oxidation state and is reactive with thiolate groups. Similar approaches 
may be used to lower the oxidation state of other medically useful metal 
ions for subsequent binding to thiolate groups. The type of the metal ion, 
its isotopic nature, and concentration would depend on the intended 
medical application. 
The product resulting from the methods set forth herein can be used for 
both medical applications and veterinary applications. Typically, the 
product is used in humans, but may also be used in other mammals. The 
product may be used to monitor normal or abnormal metabolic events, to 
localize normal or abnormal tissues, to localize diseases, and to bind to 
blood constituents, including blood cells, such as lymphocytes, for 
subsequent localization of diseases, infections, and abnormal tissues. The 
application and medical use of the product depends on the type of protein 
and the type of medically useful metal ion used. 
The product can be used in a variety of medical procedures including gamma 
scintigraphy, specific photon emission computerized tomography, positron 
emission tomography, and magnetic resonance imaging. The medical 
application of the product of this invention depends on the type of 
protein and the type of medically useful metal ion used. 
The invention is further illustrated by the following non-limiting 
examples. 
EXAMPLE I 
The PHA-L4 isolectin was obtained from E-Y Laboratories (San Mateo, CA), 
and was determined to have a purity of 97%, as determined by HPLC. PHA-L4 
was radiolabeled with .sup.125 I, and binding of the radiolabeled material 
to lymphocyte-membrane proteins attached to a solid phase was 
demonstrated. Radioiodination was accomplished using the Iodobead method, 
following the manufacturer's instructions (Pierce Chemical, Rockford, IL). 
Table 1 contains data showing binding of PHA-L4 preparations to the solid 
phase membrane proteins and the lack of binding of a control material, 
casein. 
Binding of the radiolabeled isolectin was determined by measuring the 
percentage of the total radioactivity that bound to the solid-phase 
binding protein, as described by Rhodes, et al. Rhodes, B. A., Buckelew, 
J. M., Pant, K. D., et al, "Quality control test for immunoreactivity of 
radiolabeled antibody," BioTechniques 8:70-74, 1990. Solid-phase human 
white blood cells were prepared by separating white blood cells and fixing 
them to Kynar beads. Human plasma and casein were similarly fixed to Kynar 
beads. Briefly, .sup.125 I-labeled PHA-L4 was introduced to aliquots of 
solid-phase antigen, and the initial counts per minute measured. The 
preparation was allowed to incubate, following which it was washed 
repeatedly to remove unbound .sup.125 I-labeled PHA-L4, and the final 
counts per minute were then measured. 
TABLE 1 
______________________________________ 
SPECIFIC BINDING OF .sup.125 I-LABELED PHA-L4 
TO SOLID PHASE ANTIGEN 
Solid Phase Antigen 
Initial CPM Final CPM % Bound 
______________________________________ 
Human White Blood 
58,469 15,983 27.4% 
Cells 
Human Plasma 
45,707 4,914 10.8% 
Casein Control 
46,836 1,408 3.0% 
______________________________________ 
EXAMPLE II 
PHA-L4 is labeled using the radionuclide .sup.99m Tc. PHA-L4 is obtained as 
in Example I. The L4 isolectin will not bind .sup.99m Tc directly due to 
the lack of native cysteine in the glycoprotein molecule. However, the 
PHA-L4 glycoprotein does have an ample number of lysine-containing amino 
acid groups, which are required for attaching many bifunctional chelate 
groups. The method used is conjugation of diaminedithiol to the PHA-L4 
molecule, as is described by Lever et al. Lever, S. Z., Baidoo, K. E., 
Kramer, A. V., Burns, H. D., "Synthesis of a novel bifunctional chelate 
designed for labeling proteins with technetium-99m," Tetrahedron Lett 
29:3219-3222, 1988. The conjugated PHA-L4 is then labeled with .sup.99m 
Tc, using the method described by Baidoo, et al. Baidoo, K. E., Scheffel, 
U., Lever, S. Z., .sup.99m Tc labeling of proteins: Initial evaluation of 
a novel diaminedithiol bifunctional chelating agent,"Cancer Res (Supp) 
50:799s-803s, 1990. 
EXAMPLE III 
PHA-L4 is labeled by any means known in the art, including labeled with 
.sup.99m Tc by the method in Example II. BALB/c mice, male or female, 
weighing between 18-25 grams, are injected in the right thigh, either with 
40 .mu.l turpentine or with 100 .mu.l tissue culture medium containing 
approximately 5.times.10.sup.8 E. coli (ATC-25922) and 5.times.10.sup.8 
Enterococci (ATC-29212). The organisms are grown and their numbers 
estimated by McFarlen assay in the microbiology laboratory. Forty-eight 
hours after receiving the injection, a gross swelling, approximately 0.5 
cm in diameter, appears in the thigh. 
Either .sup.111 In oxine-labeled cells or .sup.99m Tc-PHA-L4-labeled cells 
are prepared by harvesting blood from the heart of 10 anesthetized mice. 
The lymphocytes are separated, labeled, and resuspended in the mouse 
plasma. When labeling with .sup.111 In oxine, standard procedures are 
used. To label the lymphocytes with either PHA-L4 or .sup.99m Tc-PHA-L4, 
the isolectin is added to the lymphocyte/plasma suspension and allowed to 
incubate for 1 hour at 37.degree. C. PHA binds rapidly to lymphocytes, and 
this binding appears to be non-reversible after a 1-hour incubation. 
Lindahl-Kiessling, K. L., "Mechanism of phytohemagglutinin (PHA) action. 
V. PHA compared with concanavalin A (Con A)," Exp Cell Res 70:17-26, 1972. 
The cells are then centrifuged, the supernatant removed, and the cells 
resuspended in plasma. At an initial dosage of 10 .mu.g of isolectin per 
10.sup.7 cells per ml of plasma, the cells are labeled by high affinity 
bonding that apparently connects the PHA to two CD3 receptors and thus, 
avoids crosslinking the PHA between two T-cells, which can lead to 
leukoagglutination. 
The labeled cells are administered through the tail vein to groups of five 
mice, in which inflammation had been induced 48 hours earlier. 
Approximately 50 .mu.Ci of each radionuclide is injected into each animal. 
The radioactivity in each syringe is measured in a Capintech CRC-10 dose 
calibrator before and after injection, and recorded. At 4 or 24 hours post 
injection, the animals are sacrificed by placing them in an airtight jar 
containing absorbent paper saturated with halothane. The animals are 
imaged with a gamma camera equipped with a pinhole, collimator, and a 
Microdot imager. Tissue can also be dissected and weighed, and the 
concomitant radioactivity counted with an energy-calibrated, gamma 
counter. Appropriate standards prepared at the time of injection will 
allow determination of the total cpm received by each animal for each of 
the two isotopes. Corrections for decay and cross-talk can be made, and 
the result expressed as a percentage of administered dose per gram of 
tissue. 
EXAMPLE IV 
PHA-L4 is labeled by any means known in the art, including labeled with 
.sup.99m Tc by the method in Example II. It is then used as a diagnostic 
radiopharmaceutical for imaging of chronic infections such as 
osteomyelitis and granulomatous diseases. 
EXAMPLE V 
Cyclic anhydride of diethylenetriaminepentaacetic acid (DTPA) (Sigma) is 
coupled to PHA-L4. Sodium pertechnetate is added to a reducing agent, such 
as stannous chloride or dithionite, and allowed to incubate for ten 
minutes under nitrogen atmosphere. The reduced .sup.99m Tc solution is 
then added to DTPA coupled PHA-L4. Following an incubation, the PHA-L4 can 
be separated from unbound .sup.99m Tc, and the percentage of radioactivity 
associated with the PHA-L4 determined. Similar results can be obtained 
using .sup.111 In as the radionuclide. 
The preceding examples can be repeated with similar success by substituting 
the generically or specifically described reactants and/or operating 
conditions of this invention for those in the preceding example. In 
particular, the leukostimulatory agent employed may be varied, and may 
include lectins other than PHA-L4, and may further include 
leukostimulatory antibodies; the methods of radiolabeling PHA-L4 may be 
varied; the methods of production and purification of PHA-L4 may be 
varied; different preparations equivalent to PHA-L4 may be used, including 
genetically engineered materials, and the method of application and 
imaging may be varied. The foregoing are merely illustrative, and other 
equivalent embodiments are possible and contemplated. 
Although the invention has been described with reference to these preferred 
embodiments, other embodiments can achieve the same results. Variations 
and modifications of the present invention will be obvious to those 
skilled in the art and it is intended to cover in the appended claims all 
such modifications and equivalents. The entire disclosures of all 
applications, patents, and publications cited above, and of the 
corresponding application, are hereby incorporated by reference.