Note: Descriptions are shown in the official language in which they were submitted.
This application is a divisional application of
Canadian patent application serial number 404,26g filed on
June 2, 1982.
BAC~CGRO~Tl\ID OF THE INVENTION
_ __
This invention relates to compositions and methods
capable of detecting cancer cells or malignant tumors in humans.
More particularly, this invention relates to compositions radio-
labeled with Tc-99m which, when administered to a human, will
acolyte at tumor sites producing a human chorionic gonad-
tropic (hug), hug alpha subunit, hug beta subunit or an hug-
like material or b) any other tumor associated antigen to which
an antibody molecule can be prepared to include carcinoembryonic
antigen (YEA) or the like.
The use of compositions which emit radiation at levels
which can be detected after administration to the human body are
well known. These compositions are utilized to visualize anywhere
monitor the functioning of various parts of the body or are used
diagnostically to determine the absence or presence of portico-
far tissue damage or disease. In one particular aspect of the
pa prior art, radio labeled antibodies are utilized to detect t~morshaving associated therewith carcinoemhryonic antigen (YEA). As
disclosed in U.S. Patents 3,663,684, 3,867,363 and 3,927,193,
I131 or I125 labeled antibodies to YEA are utilized to detect
tumors which produce or are associated with YEA.
- It is also well known that protein molecules can be
tagged with Tc-99m in order to form diagnostic agents. An ox-
ample of such a composition is Tc~99m labeled human serum album
min. The use of chelating agents for the radio labeling of pro-
loin molecules with transition metals such as In III and/or
Tc-99m has also been described (errs et at, Pro. Nat. cad.
g Sat., U.S.A., Vol. 11, pup 3803-3806, 1976). In addition, the
use of a chelating agent for the radio labeling of antibodies
and antibody fragments including Phoebe and Fob fragments no-
active with human mizzen has been described as potentially use-
fur for imaging of myocardial infarction [Thaw and Hazer,
"Radioimmunochemical imaging of myocardial infarction: Utile-
ration of anti cardiac mizzen antibodies". In: Tumor Iamb
The Radioimmunochemical Detection of Cancer, Ed. sortie
SOW. et at, U.S.A. (New York) in prows.
It has also been proposed to tag the antibody with
peroxides (McManus et at, Cancer Research, 36, pup 2367-3481,
September, 1976) in order to localize the antigen in malignant
tumors in vitro. Furthermore, it has been proposed to label
the Gig antibody to hug with radioactive iodine in order to
localize the antigen in human choriocarcinomas transplanted in
hamster cheek pouches (Quinines et at, 1971, Journal of Nuclear
Medicine, Vol. 12, pup 69-75). Also, it is known to utilize
anti-hCG labeled with trivium or iodine to test for cancer in
a human using ash vitro diagnostic test (U.S. Patent 4,116,776,
Dalbow et at).
Recently, it has been found that neoplastic tissues
produce and/or express on their surface chorionic gonadotropin,
chorionic gonadotropin-like material, compounds similar to
and/or identical to the alpha-chain or beta-chain of chorionic
gonadotropin or mixtures thereof, specifically to the degree
where it is considered a more general marker than either car-
cinoembryonic antigen (YEA) or alphafetoprotein (AFP),(Acevedo
e-t at, "Detection and Prevention of Cancer", Part 2, Vol. I,
HUE. ~ieburgs (ED Marcel Decker, Inc., Mew York, 1978), pup 937-
I 979) The positive identification of chorionic gonadotropin
1 in a heterogeneous group of cancer cells and its non detection
in non-cancer cells in vitro has suggested -to these authors that
the compound is a common antigen (common denominator) of every
cell with onco~enic proper-ties.
While radiola~eled egg antibodies are useful for
localizing tumors in ivy, when a radioisotope of sufficient
half-life is present, the Gig antibodies comprise the immune
globulins which tend to stay in the blood stream for many hours
following intravenous administration. This increases the dip-
10 faculty in imaging the -tumor within a reasonable time period,
since blood levels of the labeled Gig antibodies maintain a
relatively high background activity. In nuclear medicine, a
high ratio of target (tumor) to background emission is desired
to obtain an image of sufficient quality to permit detection.
With short-lived radioisotopes, such as Tc9~m (6 hour half-life),
it becomes difficult to image without the use of sophisticated
background subtraction techniques (Go1denberg et at, New Erg D
J. Med., Vol. 298, pup 1384-1388, 1978).
In previous published studies, it has also been shown
I that radioiodinated Gig antibodies specific to dioxin have a
prolonged half-life in the blood stream of rabbits and baboons
compared to radioiodinated Fob fragments of the same antibody
(Smith et at, Olin. Exp. Immunol., 36, 384-396, 1979). In
these experiments, Fob fragments of anti-digoxin antibodies
were tested to determine whether the antibody fragments had a
different biodistribution pattern than the whole Gig molecule
following intravenous administration, and whether the Fob frog
mints were less toxic than Gig.
SUMMARY OF EYE INVENTION
In accordance with this invention, radio labeled come
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positions are provided which comprise antibody fragments
[Phoebe or Phoebe to hug, hug alpha subunit, hug beta subunit, or
an hCG-like material, or to any other tumor associated molecule
to which an antibody can be made including a human melanoma
associated antigen, YEA, AFT or the like that are labeled with
Tc-99m. When using the compositions of this invention to
diagnose cancer cells in a patient, the patient is administered
the Tc~99m labeled antibody fragment [Phoebe or Fob fraction
to the tumor associated antigen. The biodistribution of the
labeled composition is monitored by external gamma scintigraphy
in order to locate cancer cells or malignant tumors. The present
invention provides substantial advantages over the prior art since
the compositions provide for hither sensitivity in detecting
cancer cells or malignant tumors of the prior art, the technique
can be performed in viva, and the compositions are more effect
live in providing imaging contrast between the tumor or cancer
cells and the blood system so that good imaging can be obtained
quickly.
DESCRIPTION OF SPECIFIC E?~lBODIMEr~TS
The term, "antibody fragment", as used herein means
Phoebe or Fob fragments. The antibody fragments are prepared
by any conventional techniques such as is shown in Example I.
Human chorionic gonadotropin hug is a molecule be-
lived to have a molecular weight ranging from about 35,000 to
38,000. hug is found in the urine and sofa of pregnant women,
in patients with trophoblastic and other tumors, in the normal
placenta, and is produced by certain cell cultures hug con-
sits of two nonequivalently bonded alpha and beta chains having
approximate molecular juts of 14,700 and 23,000 respectively.
The alpha and beta chains can be easily dissociated; however,
_ _
it has been shown that each chain is biologically inactive as
a separate entity. The amino acid sequence of the alpha chain
has been shown to have close similarity to the alpha chain of
]uteinizing hormone (Hyatt), follicle stimulating hormone (hush),
and thyroid stimulating hormone (hush). The beta chain has Sims
hilarity only -to the beta chains of luteinizing hormone and less
homology to those of follicle stimulating hormone and thyroid
stimulating hormone. The beta chain is immunologically active
in both the intact hormone and as a separate entity. Appear-
mutely 30 percent of the molecule is carbohydrate which is con
stituted by six different monosaccharides: static acid, L-fruc-
lose, D-galactose, D-mannose, N-acetylglucosamine and Nastily-
galactosamine.
The antibody fragment is labeled with technetium-99m
since technetium-99m affords improved images by scintigraphy.
In contrast to iodine-labeled antibodies, technetium-99m is
retained by the antibody by a chelation mechanism. Thus, the
reagent is formed under reducing conditions in order to minimize
or prevent the reversible reaction by which the technetium em
becomes free of the antibody fragment. The source of the tech-
netium-99m preferably is water soluble such as the alkali or
alkaline earth metal pertechnetate. The technetium can be ox-
twined as sodium pertechnetate To 99m from a conventional cage
99mTc generator. Any source of pharmaceutically acceptable
technetium-99m may be utilized in the present invention.
Anti-hCG, an-ti-hCG-beta, anti-hCG-alpha or other anti-
tumor antigen antibodies are obtained by any conventional moth-
ox such as by immunizing animals such as rabbits, sheep, goats
or other suitable species with a suitable immunogen in order to
I induce production of the antibody. Serum then is harvested from
1 the immunized animals and the specific immunoglobulins then can
be obtal~ed in sufficiently pure form such as by affinity cry-
matography, immunoprecipitation, nonimmune precipitation or the
like. In affinity chromatography, for example, an hCG-rich
fraction firs-t is isolated such as from pregnant fetal serum or
urine by conventional nonimmune precipitation or immunoprecipi-
station techniques followed by chromatography on DEAE-cellulose
followed by gel filtration on Seafood* G-100 or by another suit-
able purification technique. The hCG-rich fraction thus ox-
I twined is passed onto a column of a cyanogen halide activated orperiodate activated gel such as Sephadex*, Suffers* or eel-
lulls or another insoluble polysaccharide with carboxyl, polyp
hydroxyl or N-hyclroxylsuccinimide ester functionality in order
to chemically attach the hug by a weak covalent bond to the gel.
The serum obtained from the animal then is passed through the
column and the anti-hCG, anti-hCG-beta or anti-hCG-alpha be-
comes specifically attached to the hug or hug subunits which
comprise the corresponding antigen in the column while the
remainder of the other immunoglobulins ~non-hCG specific anti-
bodies) pass through the column. The anti-hCG, anti-hCG-beta
or anti-hCG-alpha then is recovered from the column by passing
an appropriate buffer, e.g., ammonium hydroxide solution through
the column in order to break the weak covalent bond between the
antibody and the hCG-gel matrix. The antibody can be obtained
in any conventional manner such as by elusion with solution in
any conventional manner such as by elusion with solution or buy-
for of appropriate ionic strength and phi Phoebe or Fob frog-
mints then can be produced from the antibody.
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*Trade Mark
It is to be understood that the Moe of forming
the antibodies is not critical to the present invention so long
as they are in sufficiently pure form as to render the compost-
tion immunoreactive for their respective antigens. An alter-
native method for earn the antibodies useful in the present
invention comprises the method for making antibody producing
hybridomas disclosed by Killer and Millstone (1975), Nature,
Vol. 25~, pp. ~95-~97.
The technetium-99m labeled antibody fragment is pro-
pared by acidic, basic or neutral (ligand exchange) radio label-
in techniques. In one particular and preferred aspect of this
invention, the technetium-labeled antibody fragment is obtained
by a ligand exchange process. In tilts process, a solution of
technetium (IV) is prepared by mixing a solution of technetium
such as in the form of a pertechnetate (Tc04 ) and saline with
a stuns reducing solution, e.g., stuns fluoride-acetate
having a pi between about 3 and 5.5. In this procedure, the
stuns ions reduce technetium (VII) to technetium (IV). The
reduced technetium-99m first is chelated onto the top of a
coulomb of Sephadex* G-25 (dextran cross-linked with carboxyl
functionality) by passing the aqueous solution of technetium-
99m through the column. The solution has a pi between about
5.5 and 7Ø The column then is washed with saline to Essex-
tidally remove free pertechnetate (Tc04 1 or unusual species of
technetium thereby leaving the technetium-g9m chelated or Abe
sorbed or otherwise bound to the column. A physiologic soul-
lion of the antibody fragment then is prepared with appropriate
buffer so that the resultant solution has a pi between about 6
and 9, preferably between about 7 to 8. when operating within
the pi range, denaturation of the antibody fragment is elimin-
* trade Mark
axed or minimized. The antibody fragment is then added in minimum volume -to the top of -the column where the technetium
99m/Stannous complex is burled and where it is allowed to stand
until the technetium-99m is bound to the antibody fragment
having stronger bonding sites than the column material. This
usually occurs within about 30 minutes. The column then is
washed to remove the labeled antibody fragment. Washing can be
effected with a known volume of human serum albumin diluted
with 50/50 AND (acidified citrate Dextrose) or the like followed
by a known volume of saline. In this matter, the volume of
washing saline solution containing the labeled antibody fragment
can be determined and the labeled antibody will remain on the
column or will be eluded at a rate different from that of the
labeled, immunologically intact, antibody fragment.
A second preferred method for forming technetium-99m
labeled antibody fragment comprises direct labeling of the
fragment or pretend fragments. In this method, a buffered
solution is admixed with an acidic solution of Snuck which is
a reducing agent for pertechnetate. The buffered solution can
comprise sodium and/or potassium phthalatel tart rate, gentisate,
acetate, borate or mixtures thereof having a pi of between 4.$
and 8.0, preferably about 5.5. Tart rate is utilized to maintain
the appropriate concentration of stuns ion in solution to
effect the desired solution phi The Snuck preferably is added
to the buffer as a solution with concentrated HAL. Thereafter,
the solution is neutralized such as with sodium hydroxide to
attain a pi of between about 4.5 and 8. n, preferably about 5.5.
The antibody fragment then is added to the neutralized solution
in an amount to attain a concentration of protein fragment up
to just less than that which would begin to precipitate the
'Sue
; protein fragment in the buffet being used. In order to attain
the desired degree of protein fragment labeling, the resultant
stuns ion, buffer, protein fragment solution is allowed to
incubate. For example, at room temperature, the incubation
time should be at least about I hours. Preferably at least
about 20 hours under a nitrogen or an inert gas atmosphere. If
desired, this solution can be heated moderately to reduce the
incubation time. The solution then can be either freeze-dried
and subsequently reconstituted for admixture with pertechnetate
lo or can be admixed directly with pertechnetate solution to ox-
lain the labeled fragment. If desired, the resultant radio-
labeled fragment may be further purified to separate the labeled
protein fragment from free technetium such as by chromatography
in a Sephadex* column. However, this last step is optional.
The present invention also provides a kit with which
a user can prepare the composition of this invention and admix-
inter it to a patient relatively quickly after preparation.
The kit includes each antibody or antibody fragment either in
lyophilized form, frozen or liquid of suitable ionic strength
and pi, and either containing or not containing a reducing agent.
If without the reducing agent, the antibody fragment can be ad-
mixed with a reducing solution or solid provided within the sit
and in a separate container. Representative, suitable reducing
agents are Snuck or Snuff to be dissolved or already dissolved
in an appropriate solution, such as sodium acetate/acetic acid,
acidified deionized or distilled water, or the like, such that
a reducing pi of about 3 to 8 is obtained when combined with
technetium-99m as sodium pertechnetate. Therefore, technetium-
99m as pertechnetate is either reduced in the presence of no-
during agent prior to addition of the administered antibody
* Trade Mark
_ g _
1 fragment or is reduced when added to the administered antibody
fragment containing reducing agent. The solution ox labeled
antibody fragment its then suitable for administration to a
patient.
In an alternative embodiment, the eluded labeled pro-
loin fragments can be admixed with a dilute solution of human
serum albumin, e g., 1% and passed through a bed of anion ox-
change resin yin order to remove free pertechnetate from the
labeled protein fragment thereby purifying the labeled antibody
fragment so that the preparation is substantially free of radio-
chemical contamination. If desired, these anion exchange resins
need not be part of the columns utilized for labeling but can
comprise a separate bed through which the labeled protein rag-
mint is passed.
In an alternative embodiment of this invention, the
kit can include a column of material which entraps or otherwise
binds technetium~g9m such as Sephadex*, Suffers* or cellulose.
The column of this material also can contain the reducing agent
for technetium or the reducing agent can be added thereto when
I it is desired to reduce the technetium.
The labeled antibody fragment is administered by in-
ravenous injection in a pharmaceutically acceptable saline
solution, sterile and pyrogen-free. suitable dosages are
usually between about 0.5 to 30 milkers, preferably between
about 10 and 20 milkers of technetium-99m antibody fragment
for the normal 70 kg patient. The patient then can be scanned
by convention scintigraphy within 1 hour to about 5 days after
administration of the labeled protein. Tumors are located in
those areas showing a high concentration of labeled antibody.
* Trade Mark 10
1 It should be understood that -the procedure of this
invention also can be based upon antigens other than hug or
hCG--beta which are tumor specific such as carcinoembryonic anti-
guns, alpha ~e~oprotein antigens, human melanoma associated
antigens, human sacranoma associated antigens, or other tumor
specific markers wherein the antibody is produced as described
above and the antibody fragment is radio labeled.
The following examples illustrate the present inane-
lion and are not intended to limit the same.
EXAMPLE I
Figure 1 is a schematic diagram of a typical method
for making antibody fragments.
This example illustrates the preparation of Phoebe
and Fob fragments of normal rabbit Gig. The preparation of
antibody fragments of Gig is illustrated in Figure 1. Rabbit
Gig was obtained from Capper Laboratories (Cochranville, PA.
The Gig antibody was dissolved in phosphate buffered saline at
a concentration of 10 mg/ml, and was titrated to a pi of 4.0
with glacial acetic acid. Pepsin was obtained from Worthington
Biochemical, Freehold, NJ, and was added to the Gig antibody
solution at a substrate ratio of 3 my enzyme per 100 my of Gig.
The mixture was incubated at 37~C for 4 hours. After removing
any formed precipitate via centriugation, the solution was
placed on a Sephadex* (Pharmacia, Pussycat, NJ3 G-150 column
that was 1.6 x 100 cm. Three peaks of protein were eluded from
this column: Peak I corresponded to the Ftab'~2 fragment, as
determined by molecular weight sizing and i~unoreactivit~
studies showing an absence of an Fc portion of the molecule;
peaks II and III were found to contain antibody fragments that
I did not posses immunoreactivity. The Phoebe fractions were
* Trade Mark 11 -
1 pooled and were concentrated by negative pressure dialysis to
a concentration of approximately 2 mg/ml. The concentrated
Phoebe fractions were thin exposed to pa pain obtained from
soehringer Minim (Indianapolis, IMP in the presence of 2
ETA and 10 my Sistine Hal for 24 hours at 37C. The Phoebe
to pa pain ratio was 3 my per 100 my enzyme. Following this
incubation/di~est, the solution was again placed on a G-150
Sephadex* column of 1.6 x 100 cm size, with two peaks of protein
being eluded. The first peak corresponded to undigested
Phoebe, while the second peak corresponded to the Fob fraction.
The purity of the Fob fraction was determined by polyacryla~ide
electrophoresis. In an alternative method, rabbit fragments
were prepared from rabbit Phoebe by a mild reduction with 5 my
dithiothreitol for 1 hour at room temperature. The reduction
reaction was stopped by the addition of 250 Mel iodoacetamide.
The resultant Fob' was dialyzed extensively against 0.9% sodium
chloride, and was concentrated via negative pressure dialysis.
EX~lPLE II
This example illustrates a direct method of labeling
of pretend antibody fragments, such as those obtained by the
procedures outlined in Example I. Technetium-99m is obtained
from New England Nuclear Boston .~).
To 0.4 ml of 50 my sodium-potass.. us tart rate huller
pi 5.5 (10.51 gel) is added 1.6 ml of a 50 my potassium biphthal-
ate buffer pi 5.5 (10.21 g/l adjusted with 10 N Noah). To the
resultant huller solution is added 0.02 ml of 0.5 M SnC12-HCl
(94.8 g/l gone Hal). The resultant solution is titrated back
to a Pi of 5.65- + 0.05 by adding thereto 0.02 ml of 10 N Noah plus
* Trade Mark
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I additional amounts of 1 N Noah as required to obtain the specie
fled phi To this solution is added 0.3 ml of a saline solution
of the antibody to anti-hCG ~10 my protein/ml Ox saline).
The reaction vessel is allowed to stand approximately 21 hours
at room temperature under a nitrogen atmosphere. This solution
may be freeze-dried to make a Tc-99m labeling kit. Thereafter,
0.5 ml of NaTcO4 with an activity of 0.001 to 50 mCi is added
to the fragment containing composition and allowed to stand for
one half to one hour to effect substantially complete labeling
of the fragment prior to use. The resultant product is diluted
with 1.0 ml of I human serum albumin in 0.9% Nail and then is
passed through a Sephadex* (Pharmacia, Pussycat, NJ) G-25
column pretreated with stuns biphthalate to remove free To-
99m from the labeled product.
EXAMPLE I I I
This example illustrate that Fob fragments which
have been radio labeled with Tc-9~m using the pre-tinning m trod
outlined in Example II retain their immunoreactivity, and that
the Tc-99m is incorporated into the complex that forms when a
I Tc-99m labeled Fob fragment of an antibody combines with its
antigen. In this example, the antibody Fob fragment and the
other protein reagents were obtained from Capper Laboratories
(Cochronville, PA). The antigen that was used was human Gig
(Hugo), which was labeled with I-125 using a standard sheller-
amine T and sodium metabisulfide method. The antibody Fob
fragment tested in this system was a Fob fragment of sheep anti-
human Gig. Rabbit anti-sheep Gig was used to precipitate immune
complexes. Thus, a double label radio immunoassay was employed
to test the immunoreactivity of Tc-99m labeled Fob fragments.
* Trade Mark
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1 Figure 2 shows that the radioil~lunotitration curves
obtained with control Phoebes (those that were not radio labeled
with Tc-99m) and Tc-99m labeled Phoebes are essentially identical.
This illustrates that the Tc-99m labeling does not alter the
immunoreactivity of the Fob fragment. The figure also shows
that Tc-99m is incorporated into the precipitated immune complex.
Thus, Tc-99m labeled Phoebes must ye reacting with the I-125
labeled antigen.
EXPEL IV
go This example illustrates that Phoebe fragments pro-
pared according to the methods outlined in Example I are capable
of binding to immobilized antigen. The antigen in this example
is highly purified human chorionic gonadotropin hug obtained
from Syrian Laboratories, Inc. (Rome, Italy). This hug is
coupled to cyanogen bromide activated Suffers 4B obtained from
Sigma Chemical Company (St. Louis, MO). Highly purified anti-
bodies to hug were obtained from Syrian Laboratories (Rome,
twill) and Phoebe fragments of anti-hCG were prepared according
to Example I. The purified Phoebe fragments of anti-hCG were
I radio labeled with I-125 using a standard chloramine T-sodium
metablsulfide iodination reaction. The I-125 labeled Phoebe
anti-hCG fragments were then incubated with the hCG-Sepharose*
(approximately l my of hug per ml of Suffers beads) in a pros-
plate buffered saline solution (PBS) containing 1% human serum
albumin (HA) at 37C for 20 minutes in a siliconized glass test
tube. The mixture was then washed three times with the PBS-HSA
buffer to remove all unbound I-125 Phoebe fragments. The beads
were then counted to determine the percentage of total counts
added that were bound to the hCG-Sepharose*. Non-immune sheep
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* Trade Mark
1 IT was included as a control for nonspecific binding to the
hCG-Sepharose*. Specific binding to hug was determined by
elutiny the bound antibody from -the beads using 3 ml of lo pi
3.2 guanidine. After a 20 minute incubation with this
elusion buffer at 37C, the hCG-Sepharose* was washed by
centrifugation with this huller. The beads were then counted
for residual activity, with the amount of counts eluded being
considered specific binding, less the amount of CAM of the
I-125 labeled non-immune Gig eluded with guanidine. The results
lo of this experiment are shown in Table I. The results of this
experiment showed that I-125 labeled Phoebe fragments prepared
by -the methods outlined in Example I retain their immunoreact-
ivity with hug.
I
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* Trade Mark
TABLE I
BINDING OF I-125 LABELED ANTI-hCG
Phoebe FR~GMENIrS TO hCG-SEPHAROSE
. .
Percentage of Total CAM
Added Specifically wound
Sample* t hCG-Sepharose
100 no of non-immune sheep Gig
(negative control) 1.4 %
100 no of sheep anti-hCG Gig
(positive control) OWE %
100 no of sheep an-ti-hCG Phoebe 60.5 %
50 if " " " " " 53.6 %
25 11 11 if if 11 if 60. 0 I
1;2.5 " " " " " " 57.1 %
~.25 if 11 11 11 11 . 6G. 7 %
3.13 " " " " " " 5~.1 %
Average % of all Phoebe Test Samples 60.0 + 2.66
I
* All antibodies were labeled with I-125 using a standard
chloramine T-sodium metabisulfide method
Trade Mark
EXAMPLE V
This example illustrates that antibody fragments, Fob
and Phoebe, are cleared faster than are the whole antibodies
whether labeled with radio iodine or with Tc-99m. A faster blood
clearance is essential for a To 99m labeled radio pharmaceutical
because of the short half life of the Tc-99m. The whole anti-
bodies to hug were obtained as in Example IV. Lowe Phoebe were
obtained as in Example I. The Tc-9~m labeled whole antibodies
and antibody fragments were prepared as in Example II. The
purified Phoebe fragments of anti-hCG and the whole antibodies
were radio labeled with I-125 using a standard chloramine To
sodium metabisulfide iodination reaction. The radio labeled
antibodies or antibody fragments were injected via the tail
veins of female Swiss Webster mice. The animals were subset
quaintly sacrificed, dissected and the distribution of the anti-
bodies and fragment in the various tissues was determined by
measuring the radioactivity in individual tissue samples using
a gamma scintillation counter. Data presented in Tables II and
III show that the fragments are cleared from the blood more
I rapidly than are the whole antibodies.
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pi
1 TAsLF II
BLOOD LEVERS OF To LIBELED ~ITIBODY FOLLOWING IVY.
INJECTION INTO SWISS MIFF_
Time (his.) % Injected Dose/Organ
Gig Phoebe Fob
,
1 50 ND3 12
I 2 45 ND 10
33 ND 7
24 15 ND 3
72 7 ND 1.
___ _ __ _
Values obtained after injection of approximately 100 go of
protein
Percentages are from a representative experiment and are
: 15%.
3 Not determined.
I
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1 liable III
B D LEVELS OX I -LABELED ANTIBODY FOLLOWING IVY.
INJECTION INTO SWISS MICE
Time (his.) % Injected Dose/Organ
IgGF~ab')2 Fob
1 75 I I
10 2 65 17 12
24 30 4
72 20 2 2
___ _ __
1 As in Table II.
Percentages are 15% ox the actual value used to calculate
percentage.
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