CA1186620A - Composition and method for detecting cancer with technetium labeled antibody fragments - Google Patents

Abstract

ABSTRACT OF THE DISCLOSURE
This invention discloses F(ab')2 or Fab fragments of antibodies to: a) human chorionic gonadotropin (hCG), hCG
alpha subunit, hCG beta subunit, or an hCG-like material; or b) other tumor specific or tumor associated molecules, to include carcinoembryonic antigen (CEA), alpha fetoprotein (AFP), human melanoma associated antigens, human sarcoma associated antigens or other antigens, which are radiolaheled with technetium-99m (Tc-99m). When the F(ab')2 or Fab fragments of antibody to such tumor associated antigens are injected intra-venously into a patient, the radiolabeled composition accumulates at tumor sites. The accumulation of the cancer seeking radiopharmaceutical at tumor sites permits detection by external gamma scintigraphy. Thus, the composition is useful in the monitoring, localization and detection of cancer in the body.

Description

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 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.

*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 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 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 * 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

* 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.

pi 1 TAsLF II

BLOOD LEVERS OF To LIBELED ~ITIBODY FOLLOWING IVY.
INJECTION INTO SWISS MIFF_ Time (his.) % Injected Dose/Organ Gig Phoebe Fob , 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

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 As in Table II.
Percentages are 15% ox the actual value used to calculate percentage.

Claims (18)

The embodiments of the invention in which an exclusive property or privilege is claimed are defined as follows:

1. An antibody fragment capable of being radiolabeled with technetium-99m, said antibody fragment being reactive with a tumor specific antigen.

2. A composition of matter comprising a radiolabeled antibody fragment, said radiolabeled antibody fragment being directly reactive with a tumor specific antigen, said radiolabel consisting of technetium-99m.

3. A composition of matter selected from the group consisting of technetium-99m labeled fragment of anti-human chorionic gonadotropin, technetium-99m labeled fragment of anti-human chorionic gonadotropin-beta, anti-carcinoembryonic anti-gen, anti-alpha fetoprotein, anti-human melanoma antigen, anti-human neuroblastomic antigen, anti-human sarcoma antigen and mixtures thereof.

4. A composition as claimed in claim 3 wherein said anti-body is anti-human chorionic gonadotropin.

5. A composition as claimed in claim 3 wherein said antibody is anti-human chorionic gonadotropin-beta.

6. A diagnostic kit suitable for forming a composition useful in identifying a cancer cell and/or a malignant tumor which comprises a sterile package containing an antibody frag-ment being directly reactive with a tumor specific antigen and means for mixing the contents of said sterile package with reduced technetium-99m in a physiologically acceptable aqueous solution.

7. A kit as claimed in claim 6 wherein a physiologically acceptable reducing agent useful in reducing technetium (VII) to the technetium (IV) state is admixed with said protein.

8. A kit as claimed in claim 6 wherein said antibody fragment in said sterile package is lyophilized.

9. A kit as claimed in claim 7 wherein said antibody fragment and reducing agent are lyophilized.

10. A kit as claimed in claim 6 which includes a column of material capable of binding technetium in the IV state and of releasing said technetium when contacted with a solution of said antibody fragment.

11. A kit as claimed in claim 6 which includes an ion exchange resin capable of selectively removing pertechnetate ion from a solution containing pertechnetate ion from a protein labeled with technetium-99m.

12. A diagnostic kit suitable for forming a composition useful in identifying a cancer cell and/or a malignant tumor which comprise a sterile package containing a protein compris-ing a fragment of an antibody being directly reactive with a tumor specific antigen and means for mixing the contents of said sterile package with reduced Tc-99m in a physiologically accept-able aqueous solution

13. A kit as claimed in claim 12 wherein a physiological-ly acceptable reducing agent useful in reducing technetium (VII) to the technetium (VI) state is admixed with said protein.

14. A kit as claimed in claim 12 wherein said protein and reducing agent are lyophilized.

15. A kit as claimed in claim 12 wherein said protein in said sterile package is lyophilized.

16. A kit as claimed in claim 12 which includes a column of material capable of binding technetium in the IV state and of releasing said technetium when contacted with a solution of an antibody to a tumor specific antigen.

17. A kit as claimed in claim 12 which includes an anionic exchange resin capable of selectively removing pert-echnetate ion from a solution containing pertechnetate ion from a protein labeled with technetium-99m.

18. A injectable composition, which comprises:
(a) a marker-specific fragment obtained by cleavage of an antibody specific to a cytoplasmic, intracellular or cell-surface marker substance which is provided or associated with a tumor, said fragment being radiolabeled with a pharmacological-ly inert radioisotope capable of detection using a photo-scanning device; and (b) a pharmaceutically acceptable injection vehicle.