CA1186221A - Composition and method for detecting cancer with technetium labeled antibody fragments - Google Patents

Abstract

ABSTRACT OF THE DISCLOSURE

F(ab')2 or Fab fragments of antibodies to: a) human chorionic gonadOtropin (hCG), hCG alpha subunit, hCG beta sub-unit, 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 anti-gens, human sarcoma associated antigens or other antigens, are radiolabeled with tecnetium-99m (Tc-99m). When the F(ab')2 or Fab fragments of antibody to such tumor associated antigens are injected intravenously into a patient, the radiolabeled compo-sition 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.
In an alternative composition, a double antibody approach to tumor localization using radiolabeled F(ab')2 or Fab fragments is utilized. In this approach, a tumor specific antibody in the form of IgG, F(ab')2 or Fab is first administered to a patient intravenously. Following a sufficient period of time, a second antibody in the form of F(ab')2 or Fab is administered. The second antibody is radiolabeled with Tc-99m and has the property that it is reactive with the first antibody.
This double antibody method has the advantage over a single antibody approach in that smaller tumors can be localized and detected and that the total amount of radioactive trace localized at the cancer site is increased.

Description

BACKGROUND OF THE INVENTION

This invention relates to compositions and methods capable of detectiny cancer cells or malignant tumors in humans.
More particularl~, this invention relates to compositions radio-labeled wi-th Tc-99m which, when administered to a human, will accumulate at tumor sites producing a) human chorionic gonado-tropin (hCG), hCG alpha subunit, hCG beta subunit or an hCG-like material or b) any other tumor associated antigen to which an antibody molecule can be prepared to includ~ carcinoembryonic antigen (CEA) or the like.
~ The use of compositions which emit radiation a-t levels which can be detected after administration to the human body are well known. These compositions are utilized to visualize and/or monitor the functioning of various parts of the body or are used diagnosticall~ to determine the absence or presence of parti-cu]ar tissue damage or disease. In one particular aspect of the prior art, radiolabeled antibodies are utilized to detect tumors having associated therewith carcinoembryonic antigen (CEA). As disclosed in U.S. Patents 3,663,684, 3,867,363 and 3,927,193, I131 or Il 5 labeled antiboclies to CEA are utilized to detect tumors which produce or are associated with CEA.
It is also well known that protein molecules can be tagged with Tc-99m in order to form diagnostic agents. An example of such a composition is Tc-99m labeIed human serum al~umin. The use of chelating agents for the radiolabeling of protein molecules with transition metals such as In-lll and/or Tc-99m has also been described (Meares et al, Proc. Nat. Acad.
Sci., U.S.A., Vol 11, pp 3803-~806, 1976). In addition, the use of a chelating agent for the radiolabeling of antibodies and antibody fragments including F(ab'12 and Fab fragments .

2~

1 reactive with human myosin has been described as potentially use-- ful for imaging of myocardial infarction (Khaw and Haber, "Radioimmunochemical imaging of myocardial infarction~ Utili-zation of anticardiac myosin antibodies~" In~
The_Radioimmunochemical Detection of Cancer, Ed. Burchiel, S W., et al, U.S.A. (New York) in press.) It has also been proposed to tag the antibody with peroxidase (McManus et al, Cancer Research, 36. pp; 2367-3481, September, 1976) in order to locali~e the antigen in malignant tumors in vitro. Furthermore, it has been proposed to label the IgG antibody to hCG with radioactive iodine in order to localize the antigen in human choriocarcinomas transplanted in hamster cheek pouches ~.Quinones et al, 1971, Journal of Nuclear Medic e, Vol~ 12, pp. 69-75.~ Also, it is known to utilize anti-hCG labeled with tritium or iodine to test for cancer in a human us.ing an in vitro diagnostlc test, (U.S. Patent 4,116,776, Dalbow et al~) 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 carcinoembryonic antigen (CF.A) or alpha~etoprotein (AFP), ~cevedo et all, I'Detection and Prevention of Cancer", Part 2, Vol. I, H.E. Nieburgs (ED) Marcel Dekker,.Inc., New York, 197~, pp. 937-979~. Th~ positive identification o~ chorionic gona-dotropin .in a heterogenous group of cancer cells and its non-detection in non-cancer cells in vitro has suggested to these ~uthors that the compound is a common antigen (common denominatoL) of every cell with oncogenic properties.

1 While radiolabeled IgG antibodies are useful for localizing tumors in vivo, when a radioisotope of sufficient half-like is present/ the IgG antibodies comprise the immuno-globulins which tend to stay in the blood stream for many hours following intravenous administratiorl. This increases the diffi-culty in imaging the tumor within a reasonable time period, since blood levels of the labeled IgG 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 Tc-99m (6 hour half-life~, it becomes difficult to image without the use of sophisticated background subtraction techniques (Goldenberg et al, New Eng.
J. Med., Vol~ 298, pp 1384-1388, 1973). ~-In previous published studies, it has also been shownthat radioiodinated IgG antibodies specific to digoxin have a prolonged half-life in the blood stream of rabbits and baboons compared to radioiodinated Fab fragments o~ the same antibody (Smith et al, ClinO ~. Immunol., 36~ 384-396, 1979). In .
~ these experiments, ~ab fragments of anti~digoxin antibodies were tested to determine whether the antibody fragments had a different biodistribution pattern than the whole IgG molecule following intravenous adm:inistration, and whether the Fab frag-ments were less toxic than IgG.

SUMMARY OF THE INVENTION

In accordance with this invention, radiolabeled com-positions are provided which comprise: 1~ antibody fragments (F(ab')2 or Fab) to hCG, hCG alpha subunit, hCG beta subunit, or an hCG-like material, or to any other tumor associated mole-cule to which an antibody can be made including a human melanoma 1 associated antigen, CEA, ~FP or the like; and 2) antibody frag-ments (F(ab~2 and Fab) to antibodies reactive with tumor associated antigens (such as hCG, hCG alpha subunit~ hCG beta subunit, and hCG-like material, or other tumor associated molecules) that are labeled with Tc-99m. When using the com-positions of this invention to diagnose cancer cells in a patient, therefore, the treatment can be ei-ther one of two procedures.
In the first procedure (single antibody method), the patient is administered the Tc~99m labeled antibody fragment (F(ab')2 or Fab fraction~ to the tumor associated antigen. In the second procedure (multiple antibody method), the patient is first adrninistered the antibody to the tumor associated antigen (IgG, F(ab')2 or Fab fraction). After a sufficient period of time for the first antibody to accumulate at tumor sites which comprise the tumor associated antigen and for the unbound antibody to clear from the general circulation, the Tc-99m labeled second antibody fragment (either an F(ab'~2 or Fab fraction) is administered int:ravenously. The secon~ antibody has the property that it is reactive with the first antibodyO 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 ad~antages over the prior art since the compositions provide for hiyher sensitivity in detecting cancer cells or malignant tumors of the prior art, the technique can be perfo~med in vivo, and the compositions are more effective in providing imaging contrast between the tumor or cancer cells and the blood system so that good imaging can be obtained ~uickly.

DESCRIPTION OF SPECIFIC EMBODIMENTS
The term llantibody fragment", as used herein means F(ab')2 or Fab fragments. The antibody fragments ar~ prepared by any conventional techniques such as is shown in Example I.
Human chorionic gonadotropin (hCG) is a molecule believed to have a molecular weight ranging from about 35,000 to 38,000. HCG is found in the urine and sera of pregnant women, in patients with trophoblastic and other tumors, in the no:rmal placenta, and is produced by certain cell cultures. HCG con-sists oE two noncovalently bonded alpha and beta chains having approximate molecula.r weights of 14,700 and 23,000 respectively.
t~ 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 luteinizing.hormone (hLH), follicle stimulating hormone (hFSH)., and thyroid stimulating hormone (hTSH). The beta chain has similarity 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.
Approximately 30 pexcent o the molecule is caxbohydrate which . is constituted by six different monosaccharides: sialic ac.id, L-fructose, D-galactose, D-mannose, N-acetylglucosamine and N-acetylgalac-tosamine.
The antibody fragment last administered is labeled with technetium-99m since technetium-99m afords improved images by scintigraphy~ In contrast to iodine-labeled antibodies, technetium-99m is retained by *he antibody by a chelation mechanism~ Thus, the reagent is formed under reducing conditions in order to minimize or preven-t.the reversible reaction by which the technetium-99m becomes free of the antibody -fragment.

1 The source of the technetium-99m preferably is water soluble such as the alkali or alkaline earth metal pertechnetate. The technetium can be obtained as sodium pertechnetate Tc-99m from a conventional 99Mo/99mTc generator. ~ny source of pharmaceuti-cally acceptable technetium--99m may be utilized in the present invention.
Anti-hCG, anti-hCG-beta, a~ti-hCG-alpha or other anti-tumor antigen antibodies are obtained by any conventional method such as by ummunizing animals such as rabbits, sheep, goats or other suitable species with a suitable immunogen in order to induce production of the antibody. Serum then is harvested from the immunized animals and the specific immunoglobulins then can be obtained in sufficiently pure form such as by affinity chromatography, immunoprecipitation, nonimmune precipitation or the like. In affinity chromatography, for example, an hCG-rich fraction first is isolated such as from pregnant female serum or urine by conventional nonimmune precipitation or immunopre-cipitation techniques followed by chromatography on DE~E-cellu].ose followed by gel filtration on Sephadex G-100 or by ~ another suitable purification technique. The hCG-rich ~raction thus obtained is passed onto a column of a cyanogen halide activated or periodate activated gel such as Sephadex~
Sepharose or cellulose or another insoluble polysaccharide with carboxyl r polyhydroxyl or N-hydroxylsuccinimide ester functionality in order ~o chemically attach the hCG 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 becomes specifically attached to the hCG or hCG subunits which comprise the corresponding antigen 3U in the column while the remainder of the other immunoglobulins * Trade Mark - 6 -1 (non-hCG specific antibodies~ 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.~ amrnonium 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 elution with solution or buffer of appropriate ionic strength and pH. F(ab')2 or Fab fragments then can be produced from the antibody.
1~ In the multiple antibody technique, after the antibody to the tumor associated antigen i.s isolated, the antibody, hereinater referred to as a second antibody, is produced in an animal spec~ies other than the species used to produce the antibody to the tumor associated antigen, hereinafter referred to as the first antibody. Either of the two procedures described below may be utilized for antibody preparation.
1) An animal of a species different from the species in which the first antibody was produced is immunized with a nonimmune IgG fraction lnormal IgG~ of immunoglobulin from an animal species used in obtaining the first antibody in order to ~roduce a desired second antibody which binds to the first antibody or;
2~ Immune IgG fract.ion(anti-tumor associated antigen) from the animal used to produce the first antibody is adminis-tered to an animal of a different species to produce a desired second antibody which binds to its first antibody.

1 The process for obtaining the composition of this invention is illustrated by the following schematic routeo * Inject animal anti-TAA
TAA --~ species ~(lst antibody) ~ (immune IgG) species A IgG In ect animal anti A IgG
10 (nonimmune IgG) ~ species~ (2nd antibody) Produce Radiolabel ~ antibody fragment T~ Inject ¦ani~al . anti-T~a Inject~ ranimal species . ~ (lst antibody) species A ~immune IgG) ..
..... _ ~ ~

Produce ant.i anti-TAA
Radiolabel ~ antibody fragment ~____ (2nd antibody) ~ ~ _ _ . . . ~

* TAA = tumor associated antigen It is to be understood that animal species A is no-t a human. It is preferred to ut.ilize Embodiment 1 because the second antibody produced therefrom will bind specifically to any immune IgG
which immunogen (antigen) came from the animal species used to -.~f~ - 8 -7 produce the immunie IgG~ Serum containing second antibody then is harvested and the second antibody is obtained in purified form as for example by the procedures set forth above for the anti-hCG or anti-hCG-beta.
It is to be understood that the method of forming the antibodies is not critical to the present invention so long as they are in sufficiently pure form as to render the compo-sition immunoreactive for their respective antigens. An alternative method for forming the antibodies useful in the present invention comprises the method for making antibody pro-ducing hybridomas disclosed by Kohler and Milstein ~19~5), Nature, Vol. 256, pp. 495-497.
It is to be understood also that while the multiple antibody embodiment of the present invention is des.cribed above with respect to the use of two antibodies in series, the present invention is not limited to a series of only two antibodies, the second of which is radiolabeled. In order to provide higher concentrations of the radioisotope located on the tumorous cell, a series of three or more antibodies may be utiliæed, the fragment of the last of which is radiolabeled. By operating in this manner, the number of available sites associated with the tumorous cell for attachment of the radio-labeled antibody is increased greatly. The series of suitable antibodies is made as described above, with the only restrictions being that the antibodies adjacent in the series are produced from animal species different from the next adjacent antibodies in the series and that the first antibody produced in the series is anti-hCG, anti-hCG-beta, or any other antibody to a tumor associated antigen. These procedures are shown

3~ schematically as Embodiments 3 and 4~

2~.

Inject animal ¦ anti-TAA .
TAA* ._______~ species~ 7 (lst antibody) . I (immune IgG) __ Inject animal anti A IgG
species A IgG --------~ species _ ~ (2nd antibody) Inject ¦animal ¦ ¦ anti. B IgG
species B IgG _____~ species ~ (3rd antibody) A or C

Radiolabel ___~ anti~od. f ¦

-Injectanlmal anti TAA , I tl TAA _ _ _ ~ spec:ies ____~, (1 = ntibody~ n~ec anima anti anti ¦ animal ¦ Inject I anti anti TAA
ant TA~ ~ speci s~ 2nd antibody . .~ _~
Produce l antibody fragme~ adiolabel 3 0 *TAA = tumor associated an-tigen 1 The antibodies are adminis-tered parenterally to the patient in the same sequence as they are produced with the radiolabeled antibody fragment bein~ administered last. That is, the first antibody obtained as described above is administered first.
Subsequently, the second antibody is administered. The last antibody fragment .in the serles of antibodies is ra~iolabeled and will be referred to as the "last antibody fragment". I'he primary limitation on the number of antibodies that are admi-nistered is the possibility that the patient will experience undesirable side reactions to the antibodies. The possibility of undesirable side reactions occurring may be reduced by theuse of antibody fragments obtaine.d by conventional techni.ques.
The technetium-99m labeled antibody fragment is pre-pared by acidic, basic or neutral (ligand exchange) radio-labeling techniques. In one particular and preferred aspect of this invention; the technetium-labeled antibody fragment is obtained by a ligand exchange process. In this 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 stannous reducing solution, e.g., stannous fluoride-acetate having a pH between about 3 and 5.5. In this procedure, the sta:nnous ions reduce technetium ~VII) to technetium (IV). The reduced technetium-99m first is chelated onto the top of a column 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 pH
between about 5.5 and 7Ø The column then is washed with saline to essentially remove free pertechnetate ~Tc04 ) or unusual species of technetium thereby leaving the technetium-99m chelated or absorbed or otherwise bound to the column. A

* Trade Mark 7 ~

:~8~

1 physiologic solution of the antibody fragment then is prepared with appropriate buffer so that the resultant solution has a pH
between about 6 and 9~ preferably between about 7 to 8. When operating within this pH range, denaturation of the antibody fragment is eliminated or minimized. The antibody fragment is then added in a minimum volume to the top of the column where the technetium-99m/Stannous complex is bound and where it is allowed to stand until the technetium-99m is bound to the antibody fragment having stronger bonding sites than the column 1~ material. This usually occurs within about 30 minutes. The column then is washed to remove the labeled antibody fragment.
Washing can be effec-ted with a known volume of human serum albumin diluted with 50/50 ACD (acidified citrated Dextrose) or the like followed by a known volume of saline. In this manner, 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 eluted at a rate different from that of the labeled, immunologically intact, antibody fragment.
A second preferred method for forming technetium-99m labeled antibody Eragment comprises direct labeling of the fragment or pretinned fragments. In this method, a buffered solution is admixed with an acidic solution of SnC12 which is a reducing agent for pertechnetate. The buffered solution can compriselsodium and/or potassium phthalate; tartrate, gentisate, acetate, borate or mixtures thereof having a pH of between 4.5 and 8.0, preferably about 5.5. Tartrate is utilized to maintain the appropriate concentration of stannous ion in solution to effect the desired solution p~I. The SnC12 preferably is added to the buffer as a solution with concentrated HCl. Thereafter, the solution is neutralized such as with 3~
1 sodium hydroxide to attain a pH of between about 4.5 and 8~0, preferably about 5.5. The antibody fragment then is added to the neutralized solution in an amount to attain a concen-txation of protein fragment up to just less than that would beyin to precipitate the protein fragment in the buffer being used. In order to attain the desired degree of protein fragment labeling, the resultant stannous ion, buffer, protein fragment solution is allowed to incubate. For example, at room temperature, the incubation time should be at least about 15 hours, 1~ pxeferably 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 or can be admixed direct.ly with pertechnetate solution to obtain the labeled fragment. If desired, the resultant radiolabeled protein 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 provicles a kit with which a user can prepare the composi.tion of this invention and administer it to a patient relatively quickly after preparation~
The kit includes each antibody o~ antibody fragment either in lyophilized form, frozen or liquid of suitable ionic strength and p~, and either containing or not containing a xeducing agent. If without the reducing agent, the last administered antibody Eragrnent can be admixed with a reducing solution or solid provided within the k.it and in a separate container.
Representative, suitable reducing agents are SnC12 or SnF2 to be dissolved or already dissolved in an appropriate solution, such z~
1 as sodium acetate~acetic acid, acidified deionized or distilled water, or the like, such tha-t a reducing pii of about 3 to 8.0 is obtained when combined with technetium-99m as sodium per-technetate. Therefore, technetium-99m as pertechnetate is either reduced in the presence of reducing agent prior to addition of the last to be administered antibody fragment or is reduced when added to the last to be administered antibody fragment containing reducing agent. The solution of labeled antibody ~ragment is then suitable for administration to a patient.

In an alternative embodiment, the eluted labeled protein fragments can be admixed with a dilute solution of human serum albumen, e.g t 1~ and passed through a bed of anion exchange resin in order to remove free pertechnetate from the labeled protein fragment thereby purifying the labeled final anti-hody so that the preparation is substantially free of radio-chemical contamination. If desired, these anion exchange resins need not be a part of the columns utilized for labeling but can comprise a separate bed through which the labeled protein ~ra~ment is passed.
In an alternative embodimenk of this invention, the kit can include a column of material which entraps or otherwise binds technetium~9m such as Sephadex, Sepharose*or cellulose.
The column of this material also can contain the reducing agent for technetium or the reducing agent can be added thereto when it is desired to reduce the technetium.
The labeled final antibody fragment is administered by intravenous injection in a pharmaceutically acceptable saline solution, sterile and pyrogen-free. Suitable dosages are usually between about 0.5 and 30 millicuries, preferably between about 10 and 20 millicuries of technetium-99m final * Trade Mark 1 antibody frag~ent for the normal 70 kg patient. The patient then can be scanned by conventional 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 final antibody.
It should be understood that the procedure of this invention also can be based upon antigens other than hCG or hCG-beta which are tumor specific such as carcinoembryonic antigens, alpha fetoprotein antigens, human melanoma associated antigens, human sacranorna associated antigens, or other tumor specific markers wherein one or a series of antibodies are produced as described above and the last produced antibody fragment is radiolabeled.
The following examples illustrate the present invention and are not intended to limit the same.

EXAMPLE I

E'igure 1 is a schematic diagram of a typical method for makin~ antibody fragments.
This example illustrates the preparation of F(ab')2 and Fab fragments of normal rabbit IgG. The preparation of antibcdy fragments of IgG is illustrated in Figure 1. Rabbit IgG was obta~ned from Cappel Laboratories ~Cochranville, PA).
The IgG antibody was dissolved in phosphate buffered saline at a concen-tration of 10 m~/ml, and was titrated to a pH of

4.0 with ylacial acetic acid. Pepsin was obtained from Worthington Biochemicals (Millipore Corporation, Freehold, NJ), and was added to the IgG antibody solution at a substrate xatio of 3 mg enzyme per 100 mg of IgG. The mixture was incubated at 37 C for 4 hours. After removing any formed precipita-te via centrifugation, the solution was placed on a 1 Sephadex (Pharmacia, Piscataway,NJ) G-150 column that was 1.6 x 100 cm. Three peaks of protein were eluted from this column: Peak I corresponded to the F~abl)2 fragment, as determined by molecular weight sizing and immunoreactivity studies showing an absence of an Fc portion of the molecule; peaks II
and III were found to contain antibody fragments that did not possess immunoreactivity. The F(ab')2 fractions were pooled and were concentrated by negative pressure dialysis to a concentration of approximately 2 mg/ml. The concentrated F(ab')2 fractions ~ were then exposed to papain obtained from Boehringer Mannheim (Indianapolis, IN) in the presence of 2 mM EDTA and 10 mM
cysteine-HCl for 24 hours at 37C. The F(ab')2 to papain ratio was 3 mg per 100 mg enzyme. Following this incubation/digest, the solution was again placed on a G-150 Sephadex column of 1.6 x 100 cm size, with two peaks of protein being eluted. The first peak corresponded to undigested F(ab')2, while the second peak corresponded to the Fab fraction. The purity of the Fab fraction was determined by polyacrylamide electrophoresis. In an alternative method, rabbit fragments were prepared from rabbit F(ab')2 by a mild reduction with 5 mM dithiothreitol for 1 hour at room temperature. The reduction reac-tion was stopped by the addition of 250 mM iodoacetamide. The resultant Fab' was dialyzed extensively against 0.9% sodium chloride, and was concentrated via negative pressure dialysis.

EXAM_LE II

This example illustrates a direct method of labeling of pre-tinned antibody fragments, such as those obtained by the procedures outlined in Example I. Technetium-99m is obtained from New England Nuclear (Boston, M~.

* Trade Mark - 16 -~, To 0.4 ml of 50 mM sodium-potassium tartrate buffer pH 5.5 (10.51 g/l~ is added 1.6 ml of a 50 mM potassium bi-phthalate buf~er pH 5.5 (10.21 g/l adjusted with 10 N NaOH).
To the resultant buffer solution is added 0.02 ml of 0~5 M
SnC12 - HCl (94.8 g/l) conc HCl). The resultant solution is titrated back to a pH of 5.65 ~ 0.05 by adding thereto 0.02 ml of 10 N NaOH plus additional amounts of lN NaOH as required to obtain the specified pH. To this solution is added 0.3 ml of a saline solution of the antibody to anti-hCG (10 mg/protein/ml 0~9~ saline). The reaction vessel is allowed to stand approxi-mately 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 Na~c04 with an activity of 0.001 to 50 mCi is added to the ragment 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 1% human serum albumin in 0.9~ NaCl and then is passed through a Sephadex*(Pharmacia, Piscataway, N~) G-25 column pretreated with stannous biphthalate to remove free Tc-99m from the labeled product.

EXAMPLE III

This example illustrates that Fab fragments which have been radiolabeled wit`h Tc-99m using the pre-tinning method outlined in Example II retain their immunoreactivity, and that the Tc-g9m is incorporated into the complex that forms when a Tc-99m labeled Fab fragment of an antibody combines with its antigen. In this example, the antibody Fab Eragment and the other protein reagents were obtained ~rom Cappel Laboratories 3~ (Cochranville, PA)o The antigen that was used was human IgG

* Trade Mark d ~ A .; ~

1 (HuIgG), which was labeled with I-125 using a standard chlor-amine T and sodium metabisulfide method. The antibody Fab fragment tested in this system was a Fab fragment of sheep anti-human IgG. Rabbit anti-sheep IyG was used to precipitate immune complexes. Thus, a double label radioimmunoassay was e~ployed to test the immunoreactivity of Tc-99m labeled Fah fragments.
Figure 2 shows that the radioimmunotitration curves obtained with control Fab's tthose that were not radiolabeled with Tc-99m) and Tc-99m labeled Fab's are essentially identical.
This illustrates that the Tc-99m labeliny does not altex the immunoreactivity of the Fab fragment. The figure also shows that Tc-99m .is incorporated into the precipitated immune complex.
Thus, Tc-9~m labeled Fabls must be reacting with the I-125 labeled antigen.

EX~MPLE IV

This example illustrates that F(ab')2 fragments prepared a,ccordin~ to the methods outlined in Example I are capable of binding to immo~ilized antigen. The anti~en in this example is hi~hly purified human chorionic gonadotropin ~hCG~ obtained from Serono Laboratories, Inc. (Rome~ Italy).
This hCG is coupled to c~ano~en bromide activated Sepharose 4B
obtained from Sigma Chemical Company (St. Louis, M0~ hly purified antibodies to hCG were obtained ~rom Serono Laboratories (Rome, Italy) and F~ab'~2 fragments of anti-hCG were prepared accoxdin~ to Example I. The purified F(ab')2 fragments of anti-hCG were radiolabeled with I-125 using a standard chloram;ne T-sodium metabisul~ide iodination reaction. The I-125 labeled F(,ab'~2 anti hCG fragments were then incubated with the hCG~Sepharose (approximately 1 mg of hCG per ml of * Trade Mark - 18 -'~b,.' ~L~B~Z~
1 Sepharose beads) in a phosphate buffered saline solution ~PBS)containing 1% human serum albumin (HSA) at 37C for 20 minutes in a siliconized glass test tube. ThQ mixture was then washed three times with the PBS-HSA buffer to remove all unbound I-125 F(ab')2 fxagments. The beads were then counted to determine the percentage of total counts added that were bound to the hCG-~epharose. Non-immune sheep IgG was included as a control for nonspecific binding to the hCG-Sepharose. Spe~ific bindin~

to hCG was determined by eluting the bound antibody from the beads using 3 ml of l M pH 3.2 quanidine. Af-ter a 20 minute incubation with this elution buffer at 37C, -the hCG-5epharose was washed by centrifugation with this buffer. The beads were then counted for residual activity, with the amoun-t of counts eluted being considered specific binding, less the amount of CPM of the I-125 labeled non-immune IgG eluted with guanidine.
The results of this experiment are shown in Table I. The results o this experiment showed that I-125 labeled F(ab')2 fragments prepaxed by the methods outlined in Example I retain their immunoreactivity with hCG.

TABLE I

BINDING OF I--125 LABELED ANTI--hCG
F (ab')2 FRAGMENTS TO hCG-SEPHAROSE

Percentage of Total CPM
Added Specifically Bound Sample* to hCG Sepharose . _ _ . . .
lO0 ng of non-immune sheep IgG
~negative control~ l.4%

lO0 ng of sheep anti-hCG IgG
(positive control) 53.4 lO0 ng of sheep anti-hCG F(ab')2 60.5%

50 " " " " " " - 53.6 25 " " " " " " 50.0~

12.5 " " " " " " 5~.1%

6.25 " " " " " " 66.7~

3.13 " " " " " " . 57.1%
- . ... ~
Average ~ of all F(ab'~2 Test Samples 60.0 ~ 3.6 %
.. _.___ _ .. _ . . .____ _~_ ...... .. _ .. _ .... __ .. _ . _ * All antibodies were labeled with I-125 using a standard.
chloramine T-sodium metabisulfide method EXAMPLE V
This example illustrates that antibody fragments, Fab ancl F(ab')2, are cleared faster than are the whole antibodies whether labeled with radioiodine or with Tc-99m. A faster blood clearance is essential for a Tc-99m labeled radiopharmaceutical because of the short half-life of the Tc-99m. The whole an-ti-bodies to hCG were obtained as in Example IV. The F~ab')2 were obta.ined as in Example I. The Tc-99m labeled whole antibodies and antibody fragments were prepared as in Example II. The 1 purified F(ab')2 fragments of anti-hCG and the whole antibodies were radiolabeled with 1-125 using a standard chloramine T-sodium metabisulfide iodination ~eaction. The radiolabeled antibodies or antibody fragments were injected via the tail veins of female Swiss Webs-ter miceO The animals were subsequently sacrificed, dissected and the distribution of the antihodies 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
1~ show that the fragments are cleared from the blood more rapidly than are the whole antibodies.
TABI,E II

BLOOD LEVELS OF Tc-99m-LABELED ANTIBODY FOLLOWING I.V.
INJECTION INTO SWISS MICE

Time (hrs.) % Injected Dose/Organ IgG F(ab')2 Fab _ _ _ _ _ _ . _ _ ~ 24 15 ND 3 Values o~tained after injection of approximately 100 pg of protein.
Percentages are from a representative experiment and are ~ 15~. ' 3Not determined.

BLOOF LEVELS OF I -LABELED ANTIBODY FOLLOWIN~ I.V.
INJECTION INTO SWISS MICE

~ Injected Dose/Organ Time (hrs.) IgG F(ab')2 Fab ~5 10 10 2~ 30 4 As in Table II.
Percen-tages are + 15~ of the actual value used to calculate percentage.

Claims (11)

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 second antibody or second antibody fragment which is directly reactive with a tumor specific antigen, said reactivity with said second antibody, or second antibody fragment either being direct or through a series of at least one third antibody or third antibody fragment.

2. A composition of matter comprising a radiolabeled antibody fragment, said radiolabeled antibody fragment being reactive with a second antibody or second antibody fragment which is directly reactive with a tumor specific antigen, said reactivity with said second antibody, or second antibody fragment either being direct or through a series of at least one third antibody or third antibody fragment, the last of said series being directly reactive with said second antibody or antibody fragment said radiolabel consisting of technetium-99m.

3. The composition of claim 2 wherein said second antibody is anti-human chorionic gonadotropin.

4. The composition of claim 2 wherein said second antibody is anti-human chorionic gonadotropin-beta.

5. The composition of any one of claims 2, 3 or 4 wherein said radiolabeled antibody fragment is directly reactive with said antigen.

6. A diagnostic kit suitable for forming a composition useful in identifying a cancer cell and/or malignant tumor which comprises a sterile package containing an antibody fragment being reactive with a second antibody or second antibody fragment which is directly reactive with a tumor specific antigen, said reactivity with said second antibody or second antibody fragment either being direct or through a series of at least one third antibody or third antibody fragment, 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 antibody fragment.

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.