Radiation delivery to lymphoid and marrow tissues

A method of selectively delivering radiation homogeneously to lymphoid and marrow tissues in vivo including the step of administering an effective dose of a radiolabeled immunological binding partner of a T-200 antigen to a patient is disclosed. Also disclosed are immunological binding partners and compositions based thereon for carrying out the method.

FIELD OF THE INVENTION 
The present invention relates to methods and compositions for use in 
delivering radiation homogeneously to lymphoid and marrow tissue in 
patients. 
BACKGROUND OF THE INVENTION 
Before the present invention, radiation delivery in the bone marrow 
transplant setting has depended upon the clinical indication. When 
treating marrow-based diseases by a marrow transplant, irradiation has 
been delivered almost solely by external beam total body irradiation 
(TBI). Attempts to improve the rate of eradication of malignant cells by 
increasing the dose of radiation delivered have generally been limited by 
toxicity to normal organs such as lung or liver. 
Marrow transplants in which the main goal is marrow ablation (rather than 
eradication of a malignancy) have used either TBI with chemotherapy, or a 
chemotherapy-only regimen. In either case, the therapy is not delivered 
specifically to sites of disease, but is instead given systemically, and 
each type of regimen has potential toxicities to normal organs. The 
ability to avoid exposing the lung and liver to high doses of irradiation 
and/or chemotherapy, while delivering adequate doses to marrow and 
lymphoid tissue, would allow transplants for these conditions to be 
performed with less risk to the patient. 
Immunosuppression in the marrow transplant setting has generally been 
achieved by TBI/chemotherapy, or chemotherapy-only regimens, which again 
have a high rate of toxicity to normal organs. In some cases, such as 
mismatched or T-cell depleted marrow transplants, even the usual 
TBI-containing regimens frequently fail to achieve adequate 
immunosuppression and graft rejection occurs in an unacceptably high 
number of patients. The delivery of additional "total lymphoid 
irradiation" (TLI) by directed external beam radiotherapy has increased 
the degree of immunosuppression but its use has been limited by toxicity 
to neighboring tissues, particularly the mucosa of the mouth, oropharynx, 
and esophagus. 
In summary, although in general higher doses of irradiation have led to 
decreased rates of relapse and enhanced immunosuppression, this has not 
translated to improved survival because of the accompanying severe 
regimen-related toxicity when this irradiation is delivered via 
traditional, external beam sources (TBI or TLI). 
In general, most previous reported attempts to destroy malignant or 
otherwise undesirable cells with a targeted "magic bullet" approach have 
purposefully used monoclonal antibodies which are as "tumor specific" as 
possible. For example, in experimental studies of radiolabeled antibody 
therapy of lymphoma, the present inventors have tested the theoretically 
most "tumor-specific" reagent possible, which is an antibody reactive with 
the "idiotypic determinant" which is specific to the immunoglobulin 
molecule expressed only by the cells of the lymphoma itself (that is, not 
by normal lymphocytes). Alternatively, they have tested the approach of 
targeting an antigen present on most B cells, including the lymphoma 
cells. This is, in a sense, "one step less specific" than the 
anti-idiotype antibodies. In this case, the selection of a pan-B reactive 
antibody is one of convenience, in that such an antibody reacts with most 
lymphomas (and therefore need not be tailor-made for each patient), yet 
still is relatively tumor-specific; the reactivity with normal 
nonlymphomatous B cells is accepted although such cells are not the 
primary target. Other clinical trials of targeted radiotherapy using 
monoclonal antibodies have also generally employed antibodies which are as 
tumor-specific as possible, accepting nontumor reactivity as an 
unavoidable side effect. 
SUMMARY OF THE INVENTION 
A unique aspect of the invention is that it was developed starting with an 
express goal of targeting neighboring normal cells as well as the 
malignant cells, given that it is desired to treat patients whose 
malignancy is in remission, as well as those in relapse. Patients in 
remission have, by definition, too few malignant cells to be detectable by 
usual clinical means. As an example, a patient with acute lymphocytic 
leukemia is said to be "in remission" if there are fewer than 5% of cells 
in the marrow that have the appearance of blast cells. In this setting, 
targeting only the malignant cells with radiolabeled antibody would not 
allow optimum delivery of radiation to such cells if they are relatively 
isolated and are scattered among normal cells. Since the radioactivity 
from the isotope on an antibody molecule bound to a cell can be emitted in 
any direction, only a small percentage of the radioactivity from 
antibodies bound to any one cell will be delivered to that cell; thus, a 
very high number of bound antibody molecules will be required to mediate 
delivery of adequate irradiation to cause cell death. Such isolated cells 
would be much more likely to be lethally irradiated if the surrounding 
cells are also bound by radiolabeled antibody, and a lower average number 
of antibody molecules bound per cell will be required. This approach 
further allows killing of malignant cells which may not express the target 
antigen, providing they are surrounded by a significant number of cells 
which do. 
This concept led to selection herein of the T200 (CD45) antigen as a target 
for radiolabeled immunological binding partner (e.g., antibody) therapy. 
This differs from the approach used by others particularly because it is 
the most widely distributed hematopoietic antigen and therefore the least 
specific potential target for treatment of hematopoietic malignancies. 
Because the T200 antigen is widely distributed, being present on virtually 
all circulating white blood cells and on reticuloendothelial cells in lung 
and liver, one acquainted with the known distribution of this antigen 
would predict that a radiolabeled anti-T200 monoclonal antibody would 
probably result in fairly nonspecific delivery of radiation when 
administered in vivo; that is, that both the binding of the antibody to 
resident macrophages in liver and lung, as well as the pooling of 
circulating, antibody-coated leukocytes in these organs would result in 
the delivery of unacceptably high doses of irradiation to these critical 
normal organs, thus limiting the therapeutic ratio achievable. An 
unprecedented and unpredictable aspect of the observed results reported 
herein was the high therapeutic ratio achieved in preliminary studies of 
the biodistribution of anti-T200 antibodies in a primate, as can be seen 
in the Examples Section herein, particularly in Tables 1-3. Although there 
is demonstrable binding of the monoclonal antibody to liver and lung 
(i.e., levels of the specific antibody that are greater than that of the 
negative control antibody), the very high levels achievable in the 
combined target tissues of marrow, lymph nodes and spleen are several fold 
higher than even the highest level in liver, kidney, or lung. Further, 
analysis of targeted cells in nodes, spleen and marrow has shown that the 
cells which express the antigen can be saturated with the administered 
antibody. Therefore, the goal of delivery of irradiation to these sites 
where cells of hematopoietic malignancy reside is achievable with 
relatively low, and certainly acceptable, irradiation to critical normal 
organs. 
DETAILED DISCLOSURE OF THE INVENTION 
The present invention provides methods and reagents for selectively 
delivering radiation to a patient. By "selectively" is meant that the 
radiation is delivered at higher levels to lymphoid and marrow tissues 
(e.g., lymph nodes and spleen) than to other tissues (e.g., liver and 
lung). For example, in vivo testing with radiolabeled antibodies against a 
T-200 antigen has surprisingly revealed that at least approximately 3-5 
fold more radiation may be delivered to the combination of spleen, lymph 
nodes, and bone marrow than to liver, kidney, or lung. 
A convenient way of expressing selectivity in the present context is by way 
of a therapeutic ratio. As used herein "therapeutic ratio" means the ratio 
of irradiation received by target tissues to that received by the critical 
normal organ receiving the highest dose. Preferably, the therapeutic ratio 
of the compositions disclosed herein will be greater than 1, more 
preferably from about 3 to about 5. This compares with therapeutic ratios 
of essentially 1 that are achieved with available current treatment 
regimens that do not deliver irradiation specifically to target tissue. 
In general, the selectivity of radiation delivery in vivo, after 
administration of the radiolabeled immunological binding partners 
discussed herein may be determined by carrying out conventional 
biodistribution measurements after a desirable time period, conveniently 
from 1 to 48 hours or more after administration of an immunological 
binding partner (IBP) such as an antibody. A preferred method of measuring 
biodistribution of radiation and, hence, selectivity is described in 
detail in the Examples section. 
For in vivo preclinical biodistribution determination, absolute values of 
percent injected dose per gram of tissue are obtained for both target 
tissues (nodes, spleen, and less reliably, marrow) and normal organs (such 
as liver and lung). We then work "backwards" from these known values, 
correlating the gamma camera images from the same time point, and generate 
time-activity curves using the earlier gamma camera images. Dosimetry is 
then estimated from these curves and the weight of the various organs. 
In clinical studies with patients, absolute values are not obtained for all 
tissues. Instead, estimates of percent dose delivered to organs are 
generated directly from the gamma camera images using organ volumes 
obtained from CT scans, and calibrating gamma camera counts to controls of 
known radioactivity. Of the organs targeted by T-200 IBPs, the spleen 
would likely be most easily imaged in such a way that would allow reliable 
dosimetry estimates, that could then be compared to estimated dosimetry 
for liver, lung, and kidney to determine therapeutic ratio and 
"specificity." Nodes are often too small to be easily imaged. Although 
marrow is easily imaged by gamma camera, definite marrow dosimetry is the 
most difficult to estimate and would therefore be less helpful than spleen 
when trying to demonstrate specificity of irradiation delivery, requiring 
marrow biopsy to determine absolute percent injected dose per gram. 
The present invention also enables delivery of radiation homogeneously 
throughout the targeted tissues. By "homogeneous" is meant that the 
radiation is delivered in substantially equal amounts throughout the 
targeted lymphoid and marrow tissues. Homogeneity of radiation delivery 
may be detected by conventional in vivo radiation detection and 
measurement techniques. For example, gamma camera images may be employed. 
Homogeneity is often determined qualitatively by visual inspection of 
images: however, gamma camera resolution is 1 cm or greater with .sup.131 
I, so homogeneity estimates are at best crude. 
Herein, the in vivo primate data is convincing: (1) autoradiography of thin 
sections of nodes and spleen show quite homogeneous distribution of 
isotope, and (2) Fluorescein-Activated Cell Sorter (FACS) analysis of cell 
suspensions from nodes, spleen and marrow demonstrates the presence of 
bound antibody on essentially all cells expressing the target antigen, 
which is virtually a saturating amount at high antibody doses. 
The T200 antigen (described in greater detail hereinbelow) is expressed on 
all circulating leukocytes, all lymphocytes, and both myeloid and lymphoid 
precursors in the marrow. It is not expressed on erythrocytes or their 
immediate precursors, on platelets, or on megakaryocytes. Thus, a T-200 
IBP targets essentially all cells in lymph nodes and other lymphoid tissue 
(such as tonsils) and all nonerythroid cells in the spleen. As the 
majority of the marrow space is devoted to myeloid cells (the usual 
myeloid to erythroid ratio in the marrow being 2-3:1), a T-200 IBP would 
thus target more than two-thirds of the cells in the marrow space. The 
T200 antigen is expressed on virtually all lymphomas, and the majority of 
acute and chronic leukemias (both myeloid and lymphoid). 
The immunological binding partners (described in greater detail 
hereinbelow) may be administered by any conventional method suitable for 
delivery of radiolabeled protein/peptide materials. The preferred method 
is by injection. Injection may be carried out intravenously, 
intraarterially, and the like. 
Intramuscular or subcutaneous administration of T-200 IBPs would most 
likely not be desirable; these routes would result in at best very delayed 
absorption into the circulation, delivering an unacceptably high dose of 
irradiation to the site of injection and impeding access of the T-200 IBP 
to the target tissues. The preferred method of administration would be 
intravenous; the length of the infusion would be determined by the dose to 
be administered, and in some cases, the rate-related side effects such as 
those seen with the administration of murine antibodies. 
The effective dose of the T-200 IBP's to be used for a given patient will 
generally be determined by the attending physician, taking into 
consideration the nature of the condition to be treated and/or the result 
to be achieved, the body weight of the patient, the severity of the 
condition, the general condition of the patient, etc. Generally, the dose 
should be sufficient to achieve the desired selectivity of radiation 
delivery to and throughout the targeted tissues. A representative dose of 
the T-200 IBP's for this purpose will generally range from about 0.1 to 
about 10 mg/kg body weight of the patient. 
As noted above, the effective dose of the T-200 IBP is likely to vary with 
the condition treated, depending on the total body "load" of cells 
expressing the T-200 antigen. Thus, a patient with acute leukemia in 
relapse whose marrow is "packed" with leukemia blasts and who has a large 
spleen is likely to require a larger dose (approximately 1-10 mg/kg) to 
achieve an acceptable therapeutic ratio than is a patient in remission, 
who may have less than half as many total cells to bind the T-200 IBP. 
This patient, or others with "normal" marrow, may need 0.1-2 mg/kg 
(estimate) if the T-200 IBP is intact antibody. Note that these estimated 
doses refer to whole antibody; the dose required of an antibody fragment 
(e.g., an Fab) may be smaller, for the same condition, when expressed as 
mg/kg because one milligram of antibody fragments contains more molecules 
(i.e., individual T200 IBP's) than one milligram of whole (intact) 
antibody. Conversely, as the smaller antibody fragments may be cleared 
more quickly from the body, a greater number of total molecules may be 
required to achieve the same degree of saturation of T-200 antigens than 
is needed with the intact antibody molecule. 
The "dose of antibody" required does not have the same meaning as the "dose 
of radioactive isotope" required. The dose of antibody should be selected 
to provide the most optimum therapeutic ratio, which ideally is determined 
for each patient with a trace-labeled "dosimetry" infusion prior to the 
actual therapeutic dose. Once the optimum dose of antibody is established, 
the radiation dose is then determined depending on the situation, and 
manipulated by changing the number of millicuries of isotope with which 
that antibody dose is labeled. Thus, a patient who is also to receive TBI 
would receive the selected dose of antibody, labeled with the amount of 
isotope estimated to deliver "X" cGy to the normal organ (usually liver, 
lung, or kidney) receiving the highest dose of irradiation. A patient to 
be treated solely with radiolabeled T-200 IBP may receive a far higher 
dose of irradiation, by labeling the same dose of antibody with several 
fold more millicuries of isotope. 
The T-200 IBPs of the present invention may be labeled with one or several 
radioactive atoms, with or without chemical ligand groups, that are 
compatible with whole body irradiation or radiotherapy. Specific 
nonlimiting examples are radioisotopes of: iodine (e.g., .sup.131 I), 
bromine (e.g., .sup.75 Br, .sup.76 Br and .sup.77 Br), fluorine (e.g., 
.sup.18 F), astatine (e.g., .sup.211 At), samarium (e.g., .sup.153 Sm), 
holmium (e.g., .sup.150-164, 166-170 Ho), rhenium (e.g. .sup.181-184, 
186-189 Rh), and yttrium (e.g., .sup.90 Y). 
Preferably, the IBP will be labeled with 1-10 radioisotope atoms, 
particularly preferably 1-5. 
While important advances are being made in developing the technology for 
other radiolabels for monoclonal antibodies, it is believed that .sup.131 
I is the best starting place for labeling new proteins. Iodination is 
always technically achievable using routine procedures (chloramine-T, 
iodogen), and the product can be purified by standard techniques, then 
evaluated for immunointegrity. A further important advantage of .sup.131 I 
is that the same isotope is used in both the imaging studies and the 
therapeutic trials--not the case for chelate labels. Because of this, 
dosimetric calculations derived from the preliminary imaging studies will 
be valid for estimating the subsequent radiation effects of various 
therapeutic doses. The beta particle emission of .sup.131 I is a very 
appropriate therapeutic agent, since its range in tissues is up to several 
millimeters and certain nonhomogeneous deposition of antibody at the 
cellular level is to be expected. 
Also contemplated are chemical ligand groups that are made up of more than 
one atom (e.g., --R--X*, where R is a linking group such as an alkyl or 
substituted alkyl chain, at least one of which is radioactive (e.g., 
C.sub.1 -C.sub.20), and X* is a radioisotope such as one of those listed 
above). These are referred to herein as radioactive groups. Examples are: 
.sup.90 Y-DTPA .sup.131 I-TCB (tyramine cellobiose), .sup.131 I-PIP 
(paraiodophenyl), and .sup.186 Rh-N.sub.2 S.sub.2. The particular 
radioisotopes which would be preferable initially for use are .sup.131 I 
and .sup.90 Y. .sup.131 I is easy to label and can label antibody to a 
high specific activity. Its beta particle is of medium energy, and its 
photon characteristics allow it to be imaged readily. .sup.90 Y-DTPA is a 
well known chelate which is easy to use; the chelate has high tumor uptake 
and the beta particle is of medium high energy. Either of these 
radioisotopes require that the IBP be labeled just prior to use, to 
minimize damage to the IBP from the radioactivity; this would essentially 
be true of any radioisotope. 
The radioactive atoms will be covalently bound (as with .sup.131 I) to the 
T-200 IBP or will be bound via chelate to the T-200 IBP. However, it is 
also possible to label the T-200 IBP with a binding partner group such as 
biotin or avidin and add one or more radiolabeled avidin or biotin 
molecules, respectively, to the T-200 IBP prior to administration [or even 
possibly after administration] to attach the radioactive label to the IBP. 
Any standard method for radiolabeling of proteins/peptides can be employed 
to attach the radioactive group(s) to the IBP. One such method is 
exemplified hereinbelow. 
The IBP will generally be an antibody or a fragment thereof that binds to a 
T-200 antigen (defined herein). Examples of suitable IBPs are monoclonal 
and polyclonal antibodies against a T-200 antigen or epitope-containing 
fragment thereof, and Fab and other binding fragments of these monoclonal 
and polyclonal antibodies. 
Especially preferred IBP's are monoclonal antibodies reactive with a T-200 
antigen. These monoclonal antibodies are typically secreted by hybridomas 
that may be conveniently prepared by standard techniques. For example, 
monoclonal antibodies that bind to T-200 antigens are described in 
Trowbridge, U.S. Pat. No. 4,582,797. Immunological binding partners, 
specific for the T-200 antigen, synthesized by recombinant DNA engineered 
prokaryotic or eukaryotic hosts (e.g., E. coli, S. cerevisiae, and COS-1 
cells), are also suitable for practicing the invention. 
Both murine and human hybridomas are contemplated; however, murine 
hybridomas are presently preferred due to greater ease in their 
preparation. The antibodies produced by murine hybridomas react with human 
T-200 antigens and so may be used in human patients. 
Preferred monoclonal antibodies for use in conjunction with the present 
invention are the antibodies produced by the hybridoma cell lines BC8 and 
9.4. Samples of these hybridoma cell lines have been deposited before the 
filing date herein at the American Type Culture Collection, 12301 Parklawn 
Drive, Rockville, Md. 20852, U.S.A., and have been accorded the accession 
numbers HB10507 (BC8) and HB10508 (9.4). 
It is of interest to note that the antibodies produced by cell line 9.4 
have a higher avidity toward the T-200 antigen expressed by human cells 
(approximately 5.times.10.sup.9 M.sup.-1) than the antibodies produced by 
cell line BC8 (approximately 5.times.10.sup.8 M.sup.-1). Unexpectedly, in 
macaque pre-clinical work, the use of the radiolabeled lower-avidity BC8 
antibodies produces a more homogeneous distribution of radiation, e.g., in 
lymph nodes, than the higher avidity AC8 antibody. Accordingly, it may be 
preferred to employ IBP's having moderate avidity towards the T-200 
antigen (i.e., about 1.times.10.sup.8 -1.times.10.sup.9 M.sup.-1), rather 
than those having high avidity (i.e., &gt;1.times.10.sup.9 M.sup.-1), under 
circumstances where a high degree of homogeneity of radiation distribution 
is critical. 
By "T-200 antigen" is meant a family of glycoproteins that are selectively 
expressed by hematopoietic cells. The family of glycoproteins has a 
molecular weight that generally ranges from about 180 kD to 220 kD, as 
determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis 
(SDS-PAGE). The T-200 antigens are also referred to in the scientific 
literature as CD45 or leukocyte common antigens. They are expressed on 
virtually all hematopoietic cells except erythroid precursors, mature 
erythrocytes, and platelets. The IBPs against T-200 of the present 
invention preferably bind to an epitope common to all isoforms and 
therefore react with all lymphocytes, circulating leukocytes, and the 
majority of cells in the marrow. Despite this broad reactivity, T-200 does 
not appear to be expressed on any nonhematopoietic cells. Also, virtually 
all hematopoietic malignancies express the T-200 antigen. 
Generally, an intact T-200 antigen (murine or human) or mixture of antigens 
(preferably still associated with the native lymphocyte) will be used as 
the immunogen in a host to prepare immunized spleen cells for fusion and 
selection, again typically using the T-200 antigen, resulting in the 
production and isolation of hybridoma cells expressing monoclonal 
antibodies that bind to T-200 antigens. Smaller fragments containing an 
epitope region of a T-200 antigen could also be used as the immunogen. 
The patients that may be treated by administration of the radiolabeled 
immunological binding partners of the present invention are generally 
those patients that a physician has determined to be suffering from a 
condition that is treatable by radiotherapy of hematopoietic cells. The 
subject treatment is particularly useful as an adunct to whole body 
irradiation. The following disclosure details some representative 
situations in which the radiolabeled immunological binding partners could 
be used. 
A. Treatment of Diseases 
1. Acute lymphocytic leukemia 
2. Acute myeloid leukemia 
3. Chromic myelogenous leukemia 
4. Non-Hodgkin's lymphoma 
5. Hodgkin's disease 
6. Myeloma and chronic lymphocytic leukemia 
B. Marrow Ablation 
1. Thalassemia 
2. Sickle cell anemia 
C. Immunosuppression 
1. Immunosuppression prior to allogeneic or autologous marrow 
transplantation 
Both human and animal patients may be treated in accordance with the 
present invention. 
By "ablation" is meant the delivery of sufficient irradiation to the marrow 
space to destroy all preexisting hematopoietic cells, thereby resulting in 
the clearance of these cells from the space, allowing for successful 
engraftment of donor hematopoietic cells. 
By "immunosuppression" is meant the impairment of the ability of the 
patient's immune system to respond to either (a) foreign antigens such as 
those present on HLA mismatched marrow; or (b) self antigens, when such 
response is inappropriate, as in autoimmune diseases. 
By "allogeneic" is meant that the donor marrow is that of another 
individual who is not an identical twin of the recipient. 
The likely protocol would include the following steps: 
(1) radiolabled antibody administration, then 
(2) chemotherapy, then 
(3) whole body irradiation (TBI) in about 200 rad doses .times. around 6 
days, then 
(4) transporting allogeneic or autologous marrow into the patient. 
Radiolabeled antibody would be given first to allow enough time for it to 
be adequately cleared from the patient's body prior to administration of 
donor marrow (to avoid irradiating the new marrow). Physiologically 
acceptable aqueous media would be such as 0.9% saline with or without 5% 
human albumin. T-200 IBP could at some point be lyophilized but would need 
to be reconstituted prior to labeling and labeled product would be 
administered in a physiologically acceptable aqueous media.

The invention now being generally described, the same will be better 
understood by reference to certain specific examples, which are intended 
to help teach one of ordinary skill in the art how to make and use the 
invention, and are not intended to be limiting of the present invention. 
EXAMPLES 
I. Methods of .sup.131 I Labeling 
(a) Diagnostic Doses (5-10 mCi range) 
Using labeling grade .sup.131 I, we plan to iodinate the antibodies in the 
leadshielded shipping vial by the chloramine-T method, in which 
electrophilic iodination of antibody tyrosyl residues is mediated through 
chloramine-T in 0.05M phosphate buffered saline. The reaction is allowed 
to progress for 5 minutes, then is quenched by the addition of a reducing 
agent, sodium thiosulfate, and carrier sodium iodide. Separation of the 
labeled antibody product from reactants is achieved by passage of the 
reaction mixture over a Sephadex G-10 column previously washed with 10 
column volumes of sterile isotonic saline. The fractions containing the 
labeled antibody are combined, passed through a 0.22 micron filter, and 
aliquotted for quality control testing and patient infusion. The .sup.125 
I labeling of control antibody projects may be done using the same 
procedures as above. 
B. Therapeutic Doses (100-plus mCi range) 
Antibodies for therapy will be labeled by a method for high specific 
activity developed in our laboratory. The various components of our remote 
radioiodination apparatus include the .sup.131 I shipping vial, which 
becomes the reaction vial, a needle block carrying two stainless steel 
spinal needles, a charcoal trap which is vented to atmosphere inside the 
stainless steel fume hood, an eluant reservoir filled with 0.05M phosphate 
buffered saline suitable for human injection, a Sephadex G-10 purification 
column, a 2-channel peristaltic pump, a radiation detector, a series of 
four electrically driven Teflon valves, and a 0.22 .mu.m filter for 
product sterilization. The entire apparatus is shielded with 3" of leaded 
glass plus 4" of lead bricks inside a stainless steel fume hood. Remote 
controls for the valves and pumps are located outside of the hood. 
The reaction takes place in the shipping vial which contains reductant-free 
high specific activity .sup.131 I. The chloramine-T labeling reagents are 
introduced into the shipping vial through stainless steel needles 
connected to the reagent inlet line. After mechanical agitation for 5 
minutes, the reaction is terminated by the addition of a Na.sub.2 S.sub.2 
O.sub.3 quenching mixture through the same needle. Iodine vapors that 
develop during agitation are vented through the charcoal trap. Passage of 
the reaction mixture over a Sephadex G-10 column occurs when inlet valves 
are rotated and a peristaltic pump transfers the mixture from the vial to 
the top of the column. After the transfer is complete, valves are rotated 
once more to direct sterile eluant from the reservoir to the column. The 
fractions eluting from the column that contain labeled antibody are 
detected by a radiation monitor and can be collected by rotating a valve 
to send the antibody eluant through the sterilization filter, into the 
radiopharmaceutical product vial. Nonproduct is passed into a waste vial. 
After all waste products have been eluted from the column, the entire 
system is eluted with the buffer, then filled with 0.1% sodium azide 
solution. The system remains sterile and pyrogen-free for six months or 
more. 
In one year, 4.2 Curies of .sup.131 I were used in this apparatus for 
antibody labeling, with a total radiation dose to labeling personnel of 
only 210 mrem to whole body and 880 mrem to hands. 
C. Evaluation of the Final Product 
The Limulus amebocyte lysate (LAL) test is used to test a 1:10 dilution of 
the product for the absence of pyrogens. A similar sample is sent to the 
clinical microbiology laboratory for sterility testing. The 
immunoreactivity of the labeled antibody is evaluated by tests of binding 
to antigen that have been developed for each antibody being used. The 
basic principle of the tests include incubation of the product with the 
antigen, followed by separation of bound and free radioactivity. The 
immunoreactivity will be tested and will be expressed as percent bound 
radioactivity. The final product will maintain 80% or greater of its 
binding capacity. The expected binding capacity has been set previously 
for each antibody. 
Radiochemical quality is checked by cellulose acetate electrophoresis. In 
this system, we use a barbital buffer pH 8. The labeled protein remains at 
the origin, and the small molecular weight reactants travel with the 
solvent front. By our methods, the radiochemical purity is routinely &gt;98%. 
Each product will also be evaluated by HPLC using a TSK column to measure 
molecular weight profile and a hydroxyapatite column to test for 
nonspecific oxidative side reactions during iodination. If the products 
show extensive radiochemical inhomogeneity by these two HPLC methods, 
additional preparative chromatography may be required to obtain the best 
possible radiolabeled antibody. 
II. Results obtained in a Primate Model 
We have studied the biodistribution of trace .sup.131 I-labeled BC8 and a 
second murine monoclonal antibody reactive with the T-200 antigen, AC8, in 
a primate model. In this model, 5 normal preadult Macaca nemestrina male 
animals were injected with 0.5 mg/kg .sup.131 I-labeled BC8 or AC8 and at 
the same time were injected with an equal amount of .sup.125 I-labeled DT 
or 1A14 antibodies, which are isotype-matched irrelevant antibodies 
serving as a control. All animals tolerated the infusions well. The 
animals were then followed for between 48 and 96 hours, with serial scans, 
complete blood counts, node and marrow biopsies, followed by sacrifice for 
sampling of all tissues. Time-activity curves were generated for organs of 
interest, and the doses to these organs from the activity in the various 
source organs was estimated from scaling the specific absorbed fractions 
given in "Specific Absorbed Fractions of Energy at Various Ages from 
Internal Photon Sources", Cristy M., Eckerman K., ORNL/TM-8381, Oak Ridge 
National Laboratory, Oak Ridge, Tenn., 1987, and are presented below (for 
0.5 mg/kg BC8 and AC8) as rad/millicurie when delivered as .sup.131 
I-labeled monoclonal antibody. 
TABLE 1 
______________________________________ 
Dosimetry Estimate in Macaques: Anti-T-200 Antibodies 
rad/millicurie .sup.131 I-BC8 (0.5 mg/kg in Macaque) 
Organ Average Stand Dev 
______________________________________ 
Marrow 56.5 31.4 
Lymph node 126.0 36.5 
Spleen 174.3 52.0 
Liver 19.8 4.8 
Kidney 11.9 2.3 
Lung 25.2 9.0 
______________________________________ 
TABLE 2 
______________________________________ 
rad/millicurie .sup.131 I-AC8 (0.5 mg/kg Macaque) 
Organ Average Stand Dev 
______________________________________ 
Marrow 66.4 22.2 
Lymph node 40.8 2.3 
Spleen 232.0 67.9 
Liver 24.7 9.8 
Kidney 8.1 3.6 
Lung 25.8 14.8 
______________________________________ 
As can be seen, the two anti-T-200 antibodies produce somewhat different 
dosimetry results. These antibodies differ in isotype (BC8 is an IgG1, AC8 
is an IgG2a), but the isotype-matched negative control antibodies do not 
differ with respect to serum clearance. Their main difference is in 
avidity: 
TABLE 3 
______________________________________ 
Antibody Species Avidity (M.sup.-1) 
______________________________________ 
BC8 Macaque 6 .times. 10.sup.7 
BC8 Humans 5 .times. 10.sup.8 
AC8 Macaque 5 .times. 10.sup.8 
AC8 Humans nonreactive 
______________________________________ 
Therefore, the avidity of AC8 in the macaque is the same as BC8 in humans, 
suggesting that the dosimetry achievable with AC8 in the macaque may be 
most predictive of dosimetry achievable with BC8 in humans. As shown in 
Table 2, almost three times more irradiation is delivered to marrow than 
to the normal organ receiving the most irradiation (here, liver and lung 
receive approximately equal doses). 
The data presented above demonstrates the therapeutic ratio that may be 
achieved using the compositions and protocols of the present invention. 
Based on the data, it can be seen that the therapeutic ratio, as defined 
hereinabove, ranges from about 1.6 to about 2.3. This is superior to 
either the predicted result or to prior protocols. 
The examples described above are merely exemplary of various aspects of the 
present invention. Variations in the actual processes described in the 
examples will be apparent to those skilled in the art. It is therefore 
intended that the protection granted by Letters Patent hereon be limited 
only by the appended claims and equivalents thereof.