CA1323563C - Lymphographic imaging method and kit - Google Patents

Abstract

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ABSTRACT OF THE DISCLOSURE
An improved method for lymphoscintigraphy or magnetic resonance lymphography involves subtraction of a negative image produced using a gross imaging agent from a positive image produced with a specific antibody imaging agent. Another embodiment of the invention uses an antibody to lymphatic tissue as an imaging agent for lymphatics. A further embodiment uses a magnetic resonance image enhancing agent for magnetic resonance lymphography.
Reagents and kits for use in the foregoing method are also provided.

Description

1323~63 Title: LYMPHOGRAPHIC IMAGING METHOD AND KIT

Inventor: Milton David Goldenberg SPECIFICATION ' BACKGROUND OF THE INVENTION
The present invention relates to a method for imaging lymphatic structures and to a kit suitable for use therefor.
There is a need, particularly in oncology, for a method that clearly delineates lymphatic structures.
Lymphatic structures, particularly lymph nodes, drain tissue and extravascular reglons of various molecular and macromolecular substances, including antigens, infectious agents, and cells, serving as a filter as well as a part of the host organism's immunological apparatus. It is well known that certain substances with appropriate physlcal properties, when in~ected into a suitable tissue plane, are transported from the in~ection site by a drainage system and sequestered in reglonal and then more distant lymph nodes. Some of these substances, particularly colloids, are passively retained in sinusolds and actively phagocytosed by the reticuloendothelial (RE) cells within the lymph node.
When a radioisotope is incorporated in such a pharma-ceutical suitable for lymphatic accretion, the lymph system, particularly the draining lymph nodes, can be imaged with a suitable scintigraphic system.
However, when a disease process impacts upon these lymphatic structures, the image of the lymph nodes may be affected in such a manner that their form and appearance is different. For example, a cancer infiltrating a lymph node may replace a laree enough portlon of the RE tissue in the node to exclude an imaging agent, e.g., a radiocolloid, from that area of the node, resulting in a "negative" image effect.
Similar results may be obtained when the lymph node structure and functlon is compromised by lnfectious agents, e.g., bacterla, fungi, parasi~es an~ viruses.

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1323~63 However, the use of such lymphoscintigraphic methods can present problems in dlagnostic inter-pretation, since "absent" lymph nodes or "decreased uptake of radioactivity" are not in themselves diagnostic of neoplastic or other involvement of the lymph nodes. Moreover, there may be lymph nodes that appear normal in the lymphosclntigram, or even show increased radlocolloid uptake, when these nodes are found to have metastases upon micro,scopic examina-tion. Conversely, nodes with no apparent metastatic involvement may show decreased or no radlocolloid uptake. Thus, a method wlth greater specificity for lymph node involvement in cancer or in infectious diseases would be of considerable diagnostic value.
Methods of localization and therapy of tumors and infectious leslons uslng labeled antlbodies and antibody fragments whlch speclflcally blnd markers produced by or associated with tumors or infectious lesions have been disclosed, inter alia, in Hansen et al., U.S. Patent 3,927,193 and Goldenberg, U.S.
Patents 4,331,647, 4,348,376, 4,361,544, 4,468,457, 4,444,744, 4,460,459 and 4,460,561 and in related pending lapplications Canadian Serial Nos. 445,774 and 459,880.
See also DeLand et al., J. Nucl. Med., 20, 1243-50 (1979) These methods use radlolabeled antlbodles which specifically bind to markers produced by or associated wlth tumors or lnfectlous leslons, and result ln a "posltlvé" image, l.e., uptake of radloactlvlty attached to the antlbody ln the structure lnvolved wlth tumor or lnfectlous leslon and havlng the approprlate antlbody target, thus permlttlng a visuallzatlon of the lnvolved structure. Further lmprovements ln the speclflclty and resolutlon of these methods ls achleved by the use of varlous subtractlon technlques whlch are also dlsclosed ln the aforementloned references, and whlch enable back-ground, non-speclflc radloactlvlty to be distlnguished from speclflc uptake by the tumor or leslon.
Others have employed lymphosclntlgraphy to study varlous types of cancers, uslng varlous lmaglng agents. Current lymphoscintigraphlc methods employ Tc-99m antimony sulfide colloid (Tc-ASC) as the imaging agent of choice, although Tc-99m stannous phytate has also been reported as useful. See, e.g., Ege et al., Brlt. J. Radlol., 52, 124-9(1979); and Kaplan et al., J. Nucl. Med., 20, 933-7(1979).

Earlier, Au-198 colloid was used, as reported by, e.g., Hultborn et al., Acta Radiol., 43, 52(1955);
Turner-Warwick, Brlt. J. Surg., 46, 574(1959);
Vendrell-Torne et al., J. Nucl. Med., 13, 801(1972);
Robinson et al., Sur~ Forum, 28, 147(1977); Sherman et al., Am. J. Roentgenol., 64, 75(1950); and Rosse et al., Mlnerva Med., 57, 1151(1966). Intraperitoneal autologous Tc-99m-labeled erythrocytes were used in medlastinal lymphosclntigraphy to s,tudy ovarlan cancer by Kaplan et al., Br. J. Radiol., 54, 126(1981).
Tc-99m-labeled liposomes were used in axillary lymphoscintigraphy of breast cancer by Osborne et al., Int. J. Nucl. Med. Biol., 6, 75(1979). Tc-99m rhenium sulfide colloid was used in breast cancer lympho-scintigraphy by Gabelle et al., Nouv Presse Med., 10, 3067(1981). The use of Tc-ASC for lymphoscintigraphic imaging of mammary and prostatic cancers, as well as for malignant melanoma, has been reported by, e.g., Ege, Sem. Nucl. Med., 13, 26(11983): Ege, J. Urol., 127, 265-9 (1982); and Sullivan et al., Am. J.
Radiol., 137, 847-51(1981).
DeLand et al., Cancer Res., 40, 2997-3001 (1980), disclose a sclntigraphic imaging method using antl-carcinoembryonic antigen antibodies labeled with I-131. They found that the tumor marker, carclno-embryonlc antlgen (CEA), was accumulated ln lymph node metastases and also in some non-metastatic lymph nodes ln the dralnage path of proxlmal tumors, and was revealed by binding to labeled antibody.
Lymph nodes have been lmaged by magnetlc reso-nance lmaglng techniques, but not wlth the use of lmage enhanclng contrast agents, and not wlth antl-body-con~ugated lmaging agents.
It ls lmportant ln certaln clinical situations to detect the presence or absence of a particular organ, such as the ovary. Moreover, it is often necessary to determlne whether an organ ls anato-mlcally correct and whether lt has pathology, e.g., obstruction, infection, neoplasia and the llke, by a non-lnvaslve technique. It would be deslrable to have an organ imaging method uslng orean-specific imaging agents that would make it posslble to obtaln a "posl~lve" lmage Or the organ, when normal,and a defect in organ visualization if pathology is present. Such a method would provide a new approach to scintigraphic and magnetic resonance imaging of organs and tissues in the body based upon their immunologlcal speciflclty.

1323~6~
Antlbody con~ugates comprising organ-specific and tissue-specific antibodies and addends for scintigraphic detection or magnetic resonance image enhancement have not been used as organ imaging reagents.
A need continues to exist for lymphographic imaging methods which are more sensitive and specific for tumor and infectious lesion invplvement in lymphatlc structures, and for organ imaging reagents and methods with high speclflcity for differentlatlon of particular organs and tissues from surrounding structures.
OBJECTS OF THE INVENTION
One ob~ect of the present invention is to provlde a method for obtalning lymphoscintigraphlc lmages that permits higher resolution and greater speclficity for tumor or lnfectious leslon involvement with lymphatlc structures.
Another ob~eot of this invention is to provide lymphomagnetic resonance imaging methods using image enhanclng agents having organ-specific and/or tumor or lesion-specific propertles as well as gross image enhancing agents.
Yet another ob~ect of the invention is to provide methods for lymphoscintigraphy and magnetic resonance lymphQgraphy which permit convenient subtraction of other organs such as the liver and spleen.
A further ob~ect of this invention is to provide organ-specific methods and agents for scintigraphic and magnetic resonance imaging.
Still another ob~ect of the invention is to provlde a method of lymphographic imaging uslng a labeled antlbody to an antigen produced by or assoclated with a tumor or lesion in the lymphatlc structure or accreting in foci therein, wherein an early image is taken using the imaging agent as a gross imaging agent, after whioh specific uptake by antigen and clearance of non-specifically bound antlbody is permitted to proceed, followed by taking a second image of the specifically bound agent, the former image being subtracted from the latter to enhance its image quality.

1323~6~
Yet a further obJect of the invention is to provide reagents and kits suitable for use in the lymphographic imaging methods of the invention.
Upon further stud~ of the specification and appended claims, further obJects and advantages of this invention will become apparent to those skilled in the art.
SUMMARY OF THE INVENTION
These obJects can be achieved by providing a lymphographic imaging method for positlve imaging of a tumor or infectious lesion or a localized product thereof in a mammalian l~mphatic structure, comprising the steps of:
(a) parenterally inJecting a mammalian subject, at a locus and by a route providing access to said lymphatlc structure, with an amount of a gross lympho-scintigraphic imaging agent or l~mphomagnetic reso-nance image enhancing agent sufficient to permit a scintigraphic image or an enhanced magnetic resonance image of said structure to be effected;
(b) obtaining a scintigraphic image or an enhanced magnetic resonance image of said structure, at a time after inJection of said agent sufficient for said agent to accrete in said structure:
(c) simultaneousl~ or at an earlier or later time, parenterally inJecting said subJect, at the same or different locus and by the same or different route, with an antibody or antibody fragment which specific-all~ blnds a marker produced by or associated wlth a tumor or infectious lesion, said antibody/fragment being labeled with a radioisotope capable of external detection or with a magnetic resonance image enhancing agent, the amount of the labeled antibod~/fragment being sufficient to permlt a sclntlgraphlc image or an enhanced magnetic resonance image of the site or sites of speclfic uptake thereof to be obtained:
td) obtaining a scintigraphic image or an enhanced magnetlc resonance image of said site or sites, at a time after ln~ectlon of said labeled antlbody/fragment sufflclent for sald antibody/
fragment to become specifically bound to said marker in said slte or sites: and (e) subtracting the image obtained in step (b) ~rom the lmage obtalned ln step (d), to produce a reflned posltive lymphographic image of said site or sites.

In another embodiment, the invention provides a lymphographic imaging method for positive lmaging of a tumor or lnfectlous leslon or a locallzed product thereof in a mammallan lymphatic structure, comprlslng the steps of:
(a) parenterally ln~ecting a mammalian sub~ect, at a locus and by a route provlding access to said lymphatic structure, with an amount of a gross lympho-scintigraphic imaging agent or lymppomagnetic reso-nance image enhancing agent sufficient to permit a scintigraphic image or an enhanced magnetic resonance image of said structure to be effected; and (b) obtaining a scintigraphic image or an enhanced magnetic resonance image of said structure, at a time after injection of said agent sufficient for said agent to accrete in said structure;
wherein said gross imaging agent comprises an antibody/fragment which specifically binds to normal lymphatic cells or tissues, said antibody/fragment being labeled with a radioisotope or a magnetic resonance enhancing agent.
In yet another embodiment, the invention provides a lymphographic imaging method for positive imaglng of a tumor or infectious lesion or a localized product thereof in a mammalian lymphatic structure, comprising the steps of:
(a) parenterally in~ecting a mammallan sub~ect, at a locus and by a route providing access to said lymphatic structure, with an amount of a gross lympho-magnetic resonance imaging agent sufficient to permit an enhanced magnetic resonance image of said structure to be effected; and (b) obtaining an enhanced magnetic resonance image of said structure, at a time after in~ection of said imaging agent sufficient for said agent to accrete in said structure.
A further embodiment of the invention relates to scintigraphic and magnetic resonance organ imaging using antibodies that specifically bind to particular organs or tissues, and con~ugated to radioisotopes an~/or magnetic resonance lmage enhanclng agents. ,~
Still further improvements can be obtalned by uslng antlbodles to normàl lymph node structures and/or tissues as the non-specific imaging agent and subtracting the resultant image from the positive image obtained using antibodies which specifically blnd to tumors or infectious lesions.

1323~3 Another embodiment of the invention relates to a method of lymphographic imaging wherein an early image is taken using a labeled specific antibody/fragment as a gross imaging agent, and a later image is taken after specific uptake by antigen and clearance of non-specifically bound antibody has occurred, the former image being subtracted from the latter to refine its image quality.
Reagents and kits useful for practicing the methods of the invention are also provided.
DETAILED DlSCUSSION
In one methodological aspect, the present invention combines two approaches hitherto employed separately, in a way which has not been suggested in the earlier work on either technique. The work of DeLand, in collaboration with the present inventor, was related to localization of radiolabeled antibodies in tumors or accreted antigen foci of the lymphatics.
The work of others was related to lymphoscintigraphic imaging of lymphatics with gross imaging agents. The present invention relates to the hitherto unsuggested correlation and computer processing of these two images to refine the positive image of a tumor or other pathological lesion, or accreted antigen focus, revealed by specific antigen-antibody binding.
The lymphographic method of the invention can be practiced elther w~th scintigraphlc or magnetic resonance imaging agents. A combination of these imaging agents can also be used, although this requires more complex instrumentatlon and data processlng and may therefore be lmpractlcal ln most cases. The subtraction of images can be readily achieved uslng conventional software. The imaging methods of DeLand et al., Cancer Res., 40, 3046(1980), are illustrative of the computerized subtraction methods known in the art.
Ma~or areas of interest for lymphography include regional spread of neoplastic and infectious lesions of the breast, colon and rectum, prostate, ovary and testes. MaJor lymph nodes involved ln these various lesions lnclude axillary and internal mammary nodes in the chest, and the pararectal, anterior pelvic (obturator), internal iliac (hypogastric), presacral, external and common iliac, and para-aortlc nodes.

1323~63 Thus, applications where lymphographic imaging would be useful include, but are not limited to, patho-logical leslons affecting the ma~or organs of the chest, abdomen and pelvis, as well as the skin, from which the regional and, subsequently, more distant lymphatics can be involved.
Scintigraphic imaging according to the method of the invention is effected by obtaining a scintigram of the lymphatic structure of interest, using as an imaging agent a radiolabeled antibody which specific-ally blnds to a marker produced by or assoclated with a ~umor or lnfectlous lesion located ln the structure or at a locus proximal to the structure and draining into the structure, such that the antigen/marker accretes in discrete foci therein; also obtaining a scintigram of the structure using a gross imaging agent which is a radiolabeled material which accretes in the structure but which does not specifically bind to the tumor or lesion, or to an accreted antibody focus; and subtracting the latter image from the former to produce a refined positive lmage of the site or sites of localization of the labeled specific antibody within the structure.
It will be appreciated that a specific labeled antibody/fragment imaging agent can function as a gross imaging agent for early imaging of a structure, when it is diffusely accreted therein, and still function at a later time as a specific imaging agent once clearance from organ background and localiza-tion/specific antigen-antibody binding at the slte or sites of speclflc uptake by tumor, leslon or dlscrete antlgen focus has occurred. Thls forms the basis for another embodlment of the present method.
The "gross" labeled lmaglng agent used to obtain the latter sclntlgram may be a radiocolloid-type agent which is scavenged by the retlculoendothelial system tRES) and accretes ln lymphatlc structures. It may also be a radlolabeled llposome or a radlolabeled agent such as galllum cltrate, labeled bleomycln, or the like, which accretes in lymphatics. Finally, and advantageously for certain cases, it may be a new type of gross imaging agent developed especially for this invention, namely, a radiolabeled antibody which specifically binds to normal lymphatic tissues or cells, but not to tumors or lesions located therein or proximal to and draining into the structure, so that it is also dlffusely distributed in the lymph nodes and reveals the internal structure thereof.

~.J ~

~ 323~,63 Examples of gross scintigraphic imaging agents tnclude but ~r~ not 11mttod to radlocollolds, e.g., Tc-ASC, Tc-99 sulfur colloid, Tc-99 stannous phytate, Au-198 collold, Hg-197 sulfide colloid, In-111 phosphate colloid and the like, as well as the other types of agents reported in the literature, repre-sentative examples of which are disclosed herein-above. Other colloidal preparations using radio-nuclides other than Tc or Au can be used, e.g., colloidal In-111, Ru-97, Ga-67, and the like, or colloids incorporating I-131 or I-123. Such preparations are conventional and well known to the ordinary skilled artisan in this field. See, e.g., Rayudu, "Radiotracers for Medlcal Appllca- tlons, Vol.
I" (CRC Press, Boca Raton, Fla., 1983).
Radiolabeled antibodies to markers character-istic of lymphatic tissue are a new kind of gross imaging agent which are also useful in the method of the present invention. They are an example of an immunologic, organ-specific imaging agent which can be used to ascertain the location and shape of a specific organ and reveal possible abnormalities therein. Such agents are useful for imaging organs other than lymphatics, e.g., liver, spleen, pancreas, and the like, and many antibodies which specifically bind to tissues of these organs are known and/or under current investigation and development.
Organ-associated and organ-specific antibodies can be developed py immunizing a suitable animal host with certain mammalian tumors or normal organ/tissue extracts and/or cells. It is well known that use of tumors as immunogens can result in antibodies which not only react with neoplasia but also with normal tissue components which sometimes show an organ-restricted nature. ~Iistogenetic and functional difi~erences between various tlssues and organs of the body of course suggest that distinct antigens are present and identifiable. A body of scientiflc literature already exists which claims the identl-fication of organ-speclfic antlgens, either using classical lmmunization approaches or by immunlzlng with specific tumors, and this is reviewed by Goldenberg et al., Cancer Res., 36! 3455(l976), showing that such antigens are known and available.

1323~63 Similar organ- and tissue-associated and specific antigens are identifiable by hybridoma methods which produce monoclonal antibodies. One recent development is the production of human hybridoma monoclonal antibodies by securing lympho-cytes or plasma cells from patients showing certain organ-restricted autoimmune diseases, e.g., thyroid-itis, gastritis, ulcerative colitis, myositis, and the like. These antibody-producing celIs are then fused in vitro with human or murine myeloma cells and hybridomas of appropriate anti-organ and antl-tissue antibody formation are produced and propagated, using well known methods. Also, patients with specific tumor types can be used as a source of such lympho-cytes or plasma cells, or such patlents can be further immunized with such tumor cells for stimulating the production of anti-organ and anti-tissue antibodles.
Thelymphatic tissue removed is then used for fusion with suitable myeloma cells, by procedures which are by now well known and conventional ln the art.

Organ-associated and organ-specific antigens can be isolated for immunization of another species, e.g., sub-human primates, rodents, rabbits, goats, etc., by a number of methods known in the art, such as isolation of cell membranes or disruption of the cells, e.g., by centrifugation, sonication, etc., to obtain intracellular antigens. It is preferable, for these purposes, to use intracellular as opposed to surface and extracellular antigens. In this manner, organ-associated and organ-speclfic antigens can be obtained from a large number of tissues and organs of the body, including brain, thyroid, parathyroid, larynx, salivary glands, esophagus, bronchus and lungs, heart, liver, pancreas, stomach and intestines, kidney, adrenal gland, ovary, testis, uterus, prostate, etc. Of further lnterest is the differentiation of different tlssue and cellular components within an organ, such as tubular and glomerular kidney, different regions and cell types of the brain, endocrine and exocrine pancreas, etc., especially by the identification of antigens and antigen epitopes restricted to the individual cell and tissue types in question, as accomplished with polyclonal and/or hybridoma-monoclonal antibody-production methods known in the art.

1~23~3 Examples of antibodies which specifically bind to lymphatic cells and/or tissues, and which are useful as gross imaging agents when labeled with a radioisotope or magnetic resonance image enhancing agent, include the T101 murine monoclonal anti-T-cell antibody reported by Royston et al., Blood, 54~SUPP1.
1), 106a(1979): and the T200 anti-lymphoreticular cell monoclonal antibody whose specificity was reported by Hsu et al., Am. J. Pathol., 114, 387 (1984). Other antibodies to T-cells and B-cells, which can also be used for such agents, include, e.g., the B1, B2 and BA1 anti-B-cell monoclonal antibodies reported in Hsu et al., Am. J. Clin. Pathol., 80, 415 (1983), and in Hsu et al., Am. J. Pathol., 114, 387 (1984); the OKT10, A1G3, HLA-DR and ~eu 10 monoclonals reported in Hsu et al., Ibid.; and anti-lymphocyte monoclonals reported by Foon et al., Blood, 60, 1 (1982), ~eBien et al., J. Immunol., 125, 2208(1980), and Beverley et al., Eur. J. Immunol., 11, 329(1981).
The antibody may be whole IgG, IgA, IgD, IgE, IgM or a fragment such as, e.g., F(ab')2, F(ab)2, Fab', Fab or the like, including isotypes and subtypes thereof. It can be a polyclonal antibody, preferably an affinity-purified antibody from a human or an appropriate animal, e.g., a goat, rabbit, mouse or the like, or a monoclonal antibody prepared by conven-tional techniques, e.g., a murine antibod~ derived from a hybridoma produced by fusion of lymph or spleen 13235~3 cells from a mouse immunized against a lymphatic system antlgen with myeloma cells from an approprlate lmmortal cell llne.
It should be noted that mixtures of antibodies, isotypes, and immunoglobulin classes, including fragments, can be used, as can hybrid antibodies and/or antibody fragments. In particular, hybrids having both T101 and T200 specificities, or hybrids having anti-T-cell and anti-B-cell specificities, may be particularly useful as gross lymphatic imaging agents, both for scintigraphy and for magnetic resonance lymphography, depending upon the label or enhancing moiety conJugated thereto. Hybrid antibody fragments wlth dual specificities can be prepared analogously to the anti-tumor marker hybrids disclosed in U.S. Patent 4,361,544. Other techniques for preparing hybrid antibodies are disclosed in, e.g., U.S. Patents 4,474,~93 and 4,479,895, and in Milstein ct al., Immunol. Todav, 5, 299(1984).
The antibody/fragment used for the specific imaging agent can be any of the antibodies which bind to tumor-specific and/or tumor/associated markers such as those disclosed in the herein referenced U.S.
Patents and Canadian Patent Applications, including hybrid antibodies and/or fragments, as well as others which are known to the ordinary skilled artisan in this field, e.g., antibodies which bind to human T-cell lymphoma viruses (HTLV), and those which are yet to be discovered. Also useful are antibodies to markers produced-by or associated with infectious lesions of the lymphatic system, or lesions located proximal to and draining lnto lymph nodes. L~mphotroplc micro-organisms include bacteria. viruses, parasites, and the like which show a predilection for soJourn ln and involvement of lymphatic structures in the body.
Among the viruses, the HTLV famlly have a predilection for T-lymphocytes, and are involved in leukemias, lymphomas, and AIDS. The HTLV form considered etio-logic for AIDS is HTLV-III. Cytomegalovirus, EB
herpes virus, and the like, also show some predilec-tion for lymphatic structures although the site of primary infection can be other tissues, with subse-quent involvement of lymphatic tissues. However, virtually all pathogenic microoorganisms can demon-strate involvement of lymphatic tissues during passage and infection in the body.

~ ` ~,--j 1323~3 Examples of antibodies to lnfectious organisms and/or antige~s produced by or accreted b~ or in the vicinity of infectlous lesions include, e.g., anti-bodies against variola virus, yellow fever virus, arboviruses, herpes viruses, myxoviruses, entero-viruses, rabies virus, hepatitis A and B viruses, Chlamydia psittaci, Rickettsia prowazeki and other rickettsia, lymphocytic choriomenlngltis virus, Neisseria meningitidis, Nelsseria g,onorrhoeae, Corynebacterlum dlphtheriae, Clostrldium tetani, Bacillus anthracis, Yersinia pestis, Vibrio cholerae, salmonella and shigella bacterial species, staphylo-cocci specles, Reponema pallidum, leptosplral specles, Mycobacterium leprae, Mycobacterium tuberculosis, Histoplasma capsulatum, Coccidioides immitis, various streptococci, Plasmodium falciparum and other plasmodia, Toxoplasma gondii, Leishmania donovani, varlous trypanosomes, Entameba hlstolytica, Giardia lambia, Trichinella spiralis, Strongyloides ster-coralis, Antiostrongylus cantonensis, Wucheria bancrofti, Schistosoma mansoni and other schlstosomal helminths, Paragonimus westermani, echinococcal speclos, and the likc. Llstlngs of representatlve disease-causing infectious organisms to which antibodies can be developed for use in this invention are contalned in the second and subsequent editions of Davls et al, "Mlcrobiology" (Harper & Row, New York, 1973 and later), and are well known to the ordlnary skllled art worker.
Again, the antibody may be whole IgG, IgA, IgD, IgE, IgM or a fragment such as, e.g., F(ab')2, F(ab)2, Fab', Fab or the like, including isotypes and subtypes thereof. It can be a polyclonal or a monoclonal antibody/fragment, a mixture of antibodies/fragments or a hybrid. Here, where the image is produced as a result of specific antibody-antigen binding rather than non-specific uptake by the RES, it may be especially advantageous to use antibody fragments which do not have the Fc portion.
The radiolabel for both t~pes of scintigraphic imaging agents is preferably an isotope with an energy in the range of 50-500 Kev. Where more than one isotope is used for simultaneous subtraction, the two labels should be of sufficiently different energies to be separately detectable with a gamma camera having a collimator with the appropriate characteristics.
Many of the preferred radiocolloids are avail-1 ~ _ `~J
~ ., ~
13235~3 able commercially or can be prepared accordlng to conventional methods reported in the literature, lncluding the illustrative references hereinabove.
Colloids having a particular range of particle size are optimal for interstitial administration and subsequent uptake b~ the lymphatic system draining into lymph nodes of interest. A particle size of less than 25 nm, e.g., 0.1 - 25 nm, preferably 1 - 20 nm, is preferred for optimal mobilization. Control of the particle size as a function of the gelling conditions for the colloid ls conventional in the art and can be done without undue experimentation by the skilled artisan.
Commercial colloids are available with accept-able partical sizes, e.g., 198-Au colloid with a ..
particle size of 2-10 nm, 99m-Tc sulphide colloid with a high but nevertheless usable particle size over 100 nm, and 197-Hg sulphide colloid with a particle size of 10-150 nm. 99m-Tc stannous phytate is ionic, and 51-Cr and 99m-Tc human serum albumin are proteinaceous with mw 60,000.
The alternative type of gross imaging agent disclosed hereinabove, i.e., an antibody to a marker associated wlth l~mphatlc tissue, can be prepared by known methods, lf existlng antlbodies are consldered unsultable or if different or more discrimlnating specificities are desired. Generally, whole lymph cells, tissue samples and/or cell or tissue fractions, membranes, antlgen extracts or purified surface antigens are used to challenge the immune system of a suitable animal, e.g., a mouse, rabblt, hamster, goat or the like, the.antigen being rendered immunogenic by aggregation if necessary and/or by coadministration with a suitable conventional ad~uvant. Hyperimmune antiserum can be isolated and polyclonal antibodies prepared by conventional procedures. Alternatively, spleen cells can be fused with immortal myeloma cells to form hybrldoma cells produclng monoclonal anti-bodles, by what are now conventional procedures. See, e.g., the procedur~s in the above-ref,erenced Canadian Patent Application Serial No.445,774 for illustra-tive technlques. Hybrldomas uslng animal, e.g., mouse, or human myeloma cell lines and animal or human spleen or lymph cells are all known in the art, and can be made and used for the present method. See, for example, Glassy et al., "Human Monoclonal Antibodies to Human Cancers", in "Monoclonal Antibodies and Cancer", Boss et al., Eds., 163-170 (Academic Press, 1323~63 1983). The specific antisera or monoclonals are screened for specificity by methods used to screen the anti-lymphocyte clones in the references cited hereinabove, which methods are also conventional by now in thls art.
In an alternative embodiment of this approach, the gross agent can be a labeled antibody to a marker associated with a lymphatic structure, e.g., lymphatic tissues or lymphocytes, wherein the antibody also specifically binds to a marker produced by or associ-ated with liver and/or spleen tissues or components.
Among the anti-l~mphatic clones disclosed hereinabove, at least the anti-T101 antibody is also cross-reactive with spleen. Antibodies which are cross-reactive with both lymphatic tissue/cells and liver and/or spleen cells/tissue can also be prepared by well-known hybrid antibody production techniques, such as those disclosed in the above-referenced U.S. 4,331,647, 4,474,893 and 4,479,895. These would combine anti-lymph tissue antibodies with antibodies which specifically bind to liver and/or spleen.
Such antibodies can be produced using liver cells isolated from normal liver tissue obtained at autopsy. For example, mice can be immunized with such tissues for a period necessar~ to evoke anti-liver antibodies. The spleens of these mice are removed and then fused, by standard methods, with a murine m~eloma cell line sultable for hybridoma productlon. Using methods already standard in the art, monoclonal anti-body-proaucing hybridomas are selected and propagated, and those with liver-restricted or liver-associated antibody production are cloned and expanded as a source of llver organ antibodies.
Similar approaches can be used with human tumors or other normal human tissues for the production of antibodies that are organ-associated or tissue-specific. Absolute tissue specificity is not required since significant quantitative differences ordinarily suffice for operational specificit~ for imaging purposes.
It will also be appreciated that the anti-liver antibodies can be used as a llver background subtrac-tion agent when visualizing tumors in the liver.
These tumors can be of non-liver origin or of liver origin. Even if a tumor of liver orlgin has the liver organ-associated antigen, subtraction of the latter 1~23~63 can be accompllshed without missing the tumor if another liver cancer-associated antigen is used as the target for the specific antl-llver cancer antibody.
For example, antibody against alpha-i~stoprotein (AFP) can be used in combination with an antibody against normal liver organ antigen, thus refining the image of areas containing AFP in the liver.
The antibodies can be radiolabeled by a variety of methods known in the art. Many of these methods are disclosed in the above-referenced U.S. Patents andC~adi~
Patent Applications, and include direct radioiodina-tion, chelate con~ugation, direct metallation, and the like. See also, Rayudu, o~. cit.; and Childs et al., J. Nuc. Med., 26, 293(1985). Any conventional method of radiolabeling which is suitable for labeling isotopes for in vivo use will be generally suitable for labeling imaging agents according to the present invention.
The gross imaeing agent will normally be admlnistered at a slte and by means that insure that it is mobilized and taken up into the lymphatic circulation. This will vary with the system to be imaged. Multiple in~ection sites may be preferable in order to permit proper drainage to the regional lymph nodes under investigation. In some cases, injections around the circumference of a tumor or biopsg site is desired. In other cases, in~ection into a particular sheath or fossa is preferred. In~ection into the webs of the fingers or toes is a common mode used to study peripheral lymphatics.
For example, for visualization of the internal mammary lymphatics in breast cancer with Tc-ASC, the radiocolloid is in~ected into the posterior rectus sheath at the insertion of the diaphragm in the subcostal site, using about 0.5 mCi of radiocolloid in a volume usually not exceeding about 0.3 ml. This method can also be used to visualize iliopelvic lymphatics in genitourinary cancers. In patients with breast carcinoma, a unilateral inJection ls made in the subcostal site ipsilateral to the tumor, and then repeated later on the contralateral side to observe cross drainage between the ipsi-lateral and contra-lateral nodes. Imaging is effected at appropriate times after each in;ection and in~ection of the specific imaging agent is coordinated with the injections of the gross imaging agent to permit optimal visualization of the positive and negative images.

~ ~J

1323~63 Images of axillary, subclavian and supra-clavicular nodes may be obtained by injecting the imaging reagents into the medial surface of the upper arms (lpsilateral and contralateral) of patients with breast cancer.
Another approach is to inJect about 0.1 - 0.5 mCi of Tc-ASC around the areola tissue of the breasts bilaterally, and then image the axilla. In addition to periareolar inJection, interdigital administration of radiocolloid may be used for visualization of axillary lymphatics (see, DeLand et al., 1980, loc.
cit.). Combined interdigital and periareolar admin-istration of radiocolloid can provide increased accuracy to demonstrate decreased uptake in affected axillary nodes. Intratumoral lnJection of, e.g., Tc-99m rhenium colloid has been performed ln patients with breast cancer and is a useful mode of adminis-tration for certain cases.
For lymphoscintigraphy of genitourinary cancers or lesions, bilateral deep perianal inJection of radiocolloid and specific imaging agent into the ischiorectal fossa is effective. For example, the patient can be placed in the lithotomy position and about 1 mCi of Tc-ASC in a volume less than about 0.3 ml is introduced bllaterally into the ischiorectal fossa, e.g., with a 22 gauge needle, to a depth of about 1.5 lnches Just lateral to the anal margln, at the 9 and 3 o'clock positions. The patient may also lie on the side if achieving the lithotomy position is not possIble. Subcutaneous dorsal pedal inJection of about 1 mCi of Tc-ASC and/or specific imaging agent may be made, e.g., using a 27 gauge half-inch needle in the first interdigital spaces bilaterally.
In certain cases, such as testicular or prostatic cancer or some cases of rectal carcinoma, intratumoral or peritumoral inJection of imaging agents can be effective.
The cross-reactive agent is preferably lnJected by a systomlc routo, o.g., lntravonously, ln~raarterl-ally, intramuscularly or subcutaneously, or by a combina~lon of systemic and lntralymphatlc routes insu!~ln~ lts accrotlon ln both the lymphatlo structure of interest and the liver and/or spleen. This technique permits subtraction of the liver and/or spleen which can further refine the image of the desired lymphatic structure. Another advantage of ~` ~
,,f 1323~3 this approach is its utility in reducing repositioning errors in sequential imaging wherein a patient is imaged in multiple sessions. The organ image can be used to correlate and superimpose temporally discrete images by computer matching of the organ image from the separate sessions.
Volumes of colloid preparations are normally about 0.1 - 2.0 ml, preferably abou~ 0.2 - 1.0 ml, per in~ection site, but this can vary depending on the site and the number of inJections. Volumes of labeled antibody gross imaging agent, normally in sterile phosphate-buffered saline (PBS) solution or sterile mineral oil suspension, will normally vary somewhat depending upon the site, the concentration and activity of the preparation, and the number of inJections.
Activity of the gross agent will normally be in the range of about 0.1 - 2.5, preferably about 0.25 -1.5, mCi per inJection for a Tc-99m-labeled agent.
Using Tc-ASC, doses of 0.25 - 1.0 mCi per inJection are given for normal in~ections. It will be appreciated that the activity will vary for other radioisotopes, depending upon their half-lives, thelr lmaging characteristics, i.e., energy ranges, emlsslon lntenslties, scatter and the like, the stabillty of the labeled agent, especially antibody conJugates, thelr rate of transport to the lymph nodes, their dlstribution and clearance, and the tlme at whlch imaglng is to be done. AdJustment of these parameters wlll be conventlonal for the ordlnary skilled cllnlcian.
Imaglng ls normally effected up to about 6 hours, more preferably at about 2 - 4 hours after lnJectlon of the gross lmaging agent, to obtaln the "negatlve" image of the lymphatic structure. Imaging of the locallzed specific imaging agent is normally effected at about 12 - 48 hours, preferably at least about 24 hrs post-lnJection, in order for the non-specifically bound antibody to clear the node. If too much of the specific agent enters the circulation, conventional subtraction agents, e.gO, 99m-pertech-netate and Tc-99m-HSA can be used to normalize.
Alternatively, second antibody, e.g., rabbit or goat anti-mouse IgG, can be inJected i.v. to enhance clearance of the specific antibody, as disclosed in Canadian Applicatlon ~erlal NO . 459 . ~80 .

1323~63 Timlng of the in~ections of gross and speclfic imaging agents will depend upon the types of agents used and the drainage patterns to the nodes of interest. Normally, it will take the specific agent a longer time to localize, and for the non-localized agent to clear the nodes, than the time required before imaging can be effected with the gross imaging agent. Thus, if it is desired to image both agents at about the same time, the specific imaging agent may need to be in~ected well before the gross agent.
DeLand et al., 1980, loc. cit., reported imaging at between about 6 and 48 hours post-in~ection for breast cancer cases, where I-131-labeled anti-CEA antibody was injected in the webs of the fingers and feet.
Combination of this procedure, according to the invention, with interdigital in~ection of Tc-ASC is advantageously effected by in~ection of the colloid about 20 - 36 hours after in~ection of the labeled antibody, and imaging of the axillary, subclavian and supraclavicular nodes about 2 hours later, using a collimator which permits separate acquisition of the I-131 and Tc-99m radiation.
It is generally preferred to effect imaging of both the gross and specific agents at the same time, using separately detectable radionuclides. This avoids the errors associated with repositioning the patient andlor realigning the images b~ computer.
Consequently, the choice of label for the gross and specific imaging agents and the activities thereof will take into co~sideration the time intervals for imaging. The specific antibody imaging agent normally will have a label with at least as long a half-life as the gross agent. In the earlier example hereinabove, the antibody is labeled with I-131, and the gross agent has a Tc-99m ~abel. The antibody could be labeled wlth In-111 and the gross agent wlth Ga-67, both of which have about the same half-life of about 2.5 days. Other pairs of compatible radionuclides for use in labeling the specific and gross imaging agents are disclosed in, e.g., the above-referenced U.S.
Patent 4,444,744.
In another alternative embodiment of the inven-tion, a labeled specific antibod~ is administered by a route and mode which ensures accretion in a lymph node to be imaged, an early image is taken when the ma~or portion of the antibody is grossly accreted in the lymph node, e.g., after 3 - 6 hours, and a later image is taken after the ma~or portion of non-localized ~J

1323~63 antibody has cleared and the ma~or portion, e.g., at least 50~, preferably at least about 70~, and more preferably at least about 90~, of the labeled anti-bod~/fragment remaining in the lymph node has been specifically bound by antigen at discrete sites in the structure. The earlier image is then subtracted by computer processing from the later image to generate a refined positive image of the structure.
The scintigram is normally taken by a gamma imaging camera having one or more windows for detec-tion of energies in the 50-500 keV range. Use of radioisotopes with high enough energy beta or positron emissions would entail use of imaging cameras with the appropriate detectors, all of which are conventional in the art.
The scintigraphic data are stored in a computer for later processing. Subtraction of the "negative"
image obtained with the gross imaging agent sharpens and refines the "positive" image obtained with the specific, locallzed labeled antibody . Subtraction is effected by the method of De~and et al., o~. clt., or v~rlant~ thos-oor~ accordlng to well-known techniques of data processing, normally lnvolving pixel-by-pixel subtraction of normalized values of counts for each channel of the detector, optlonally with correction for the counting efflciency of each channel for the radionucllde label detected thereln, and conversion of the subtracted values to an output signal to a mono-chrome or color screen. Where cross-reactive anti-bodies are used as the gross imaeing agent, computer subtraction of the image of the cross-reactive organ is also effected to further resolve the posltlve lmage of the localized antibody site or sites.
If no tumor or leslon ls present in the struc-ture, but marker accretes there by drainage from a proximal tumor or lnfectlon, the marker can accrete ln discrete focl wlthln the lymph nodes in the drainage path. This can be visualized using the present method, since the gross imaging agent wlll still en-able subtraction of areas of only diffuse accretion.
The diagnostic significance of such foci of antlgen accretion may be difficult to evaluate, and to distinguish from small metastases, but this problem is common to earlier methods and must be resolved by correlation of imaging data with other diagnostic results. It will be recognized that use of only gross imaging agents fails to reveal such antigen localiza-1323~3 tion, i.e., foci of antigen accretion, which often suggest eventual invasion of tumor cells and also reveal tumor drainage pathways.
Another important appllcatlon of the organ- or tissue-speclfic or organ- or tissue-associated anti-bodies disclosed hereinabove is for normal organ scintigraphy and mri. In thls case, a suitably radio-labeled antibody/fragment or an antibody/fragment bearing a mr image enhancing agent is administered with the lntention of obtaining a "posltlve" lmage of the organ, when normal, and a defect in organ visual-ization if pathology is present. This provides a new approach to organ and tissue-speclflc nuclear and magnetic resonance imaging of organs and tissues in the body, based upon their immunological specificity.
It will be understood that the inventlon is not limited to use of known antibodies or markers, but can be practiced with antibodies to any marker produced by or associated wlth a tumor or other pathological lesion.
Magnetic resonance imaging (mri) ls effected in an analogous manner to sclntigraphic imaging except that the imaging agents will contain magnetic resonance (mr) enhancing species rather than radio-isotopes. It will be appreciated that the magnetic resonance phenomenon operates on a different principle from sclntlgraphy. Normally, the slgnal generated is correlated with the relaxation tlmes of the magnetic moments of protons in the nuclei of the hydrogen atoms of water molecules in the region to be imaged. The magnetic resonance image enhancing agent acts by increasing the rate of relaxatlon, thereby increasing the contrast between watsr molecules ln the region where the lmaglng agent accretes and water molecules elsewhere ln the body. However, the effect of the agent ls to decrease both T1 and T2, the former resultlng ln greater contrast whlle the latter results ln lesser contrast. Accordlngly, the phenomenon ls concentratlon-dependent, and there ls normally an optimum conoentratlon of a paramagnetic species for maximum efficacy. Thls optlmal concentration will vary with the particular agent used, the locus of imaging, the mode of imaging, i.e., spin-echo, saturation-recovery, inversion-recovery and/or various other strongly T1-dependent or T2-depend-ont imaein~ ~ochniquos, and tho compo~ltlon of the medium ln which the agent is dissolved or suspended.

1323~3 These factors, and their relative importance are known ln the art. See, e.g., Pykett, Scientific American, 246, 78(1982); Runge et al., Am. J. Radiol., 141, 1209(1983).
Again, the gross agent can be a colloid or a labeled antibody to a normal component of lymphatic structures, labeled with a paramagnetlc ion or radical which can significantly alter the relaxation time of protons in water molecules in its vicinity. It is also possible to use an agent containing a high con-centration of atoms of an element other than hydrogen, having a strong nuclear magnetic moment which is detectable by an nmr detector, e.g., Fluorine-19 and the like, and which can also be accreted in a lymphatic structure in an amount sufficient for efficient nmr detection.
Examples of RE colloids useful for mri of lymphatic systems include Cd(III), Eu(III), Dy(III), PrtIII), Pa(IY), Mn(II), Cr(III), Co(III), Fe(III), Cu(II), Ni(II), Ti(III) and V(IV) colloids, colloids of other strongly paramagnetic ions, or radicals, e.g., nitroxides, and antibody con~ugates bearing paramagnetic ion chelates or radical addends. The latter will include paramagnetic con~ugates with antibodies to lymphatic structures or lymphocytes, for use as gross imaging agents, as well as conJugates with antibodies to tumor or lesion markers for use as specific imaging agents.
The specific imaging agent can use the same image enhancing agent, with mri effected at different times from the gross imaging, or a label which is separately and independently detectable with an nmr imaging camera, in the presence of the agent used for gross imaging. Examples of the latter strategy include, e.g., use of antibody con~ugates with heavy loadings of Gd(III) or Mn(II) chelates as the specific imaglng agent, where the gross imaging agent is a colloid containing a high concentration of fluorine atoms or atoms of another suitable element havlng a strong nuclear magnetlc moment, whose nuclear magnetlc resonance frequency occurs at a wldely dlfferent value from that of the hydrogen nucleus.
The mr image enhancing agent must be present in sufficient amounts to enable detection by an external camera, using magnetic field strengths which are reasonably attainable and compatible with patlent 1323~63 safety and instrumental design. The requirements for such agents are well known in the art for those agents whlch have thelr ePfect upon water molecules ln the medium, and are dlsclosed, inter alia, in Pykett, oP.
cit., and Runge et al., o~. cit.
Preparation of antibodies con~ugated to a magnetic resonance lmage enhancing agent can be effected by a varlety of methods. In order to load an antibody molecule with a large number~of paramagnetic ions, it may be necessary to react it wlth a reagent having a long tail to which are attached a multi-plicity of chelatlng groups for binding the ions.
Such a tail can be a polymer such as a polylysine, polysaccharide, or other derivatized or derivatizable chain having pendant groups to which can be bound chelating groups such as, e.g., ethylenediaminetetra-acetic acld (EDTA), dlethylenetriamlnepentaacetic acid (DTPA), porphyrins, polyamines, crown ethers, bis-thiosemicarbazones, polyoxlmes, and the like groups known to be useful for this purpose. The chelate is normally linked to the antibody by a group which enables formation of a bond to the antibody with minlmal loss of immunoreactivity and minimal aggrega-tion and/or internal cross-linking. Other, more unusual, methods and reagents for con~ugating chelates to antibodies are disclosed in copending Canadlan Patent Application Serial No. 510,508 to Hawthorne, entitled "Antibody Conjugates".

The mr scans are stored in a computer and the lmage subtraction is effected analogously to the scintigraphic data.
Reagents for use in the method of the invention include radiocolloids, radiolabeled antibodies/frag-ments which specifically bind to markers produced by or associated with tumors and infectious lesions, radiolabeled antibodies/fragments which specifically bind to lymphatic structural components, including tissues and lymphocytes, radiolabeled antibodies/
fragments which specifically bind to normal organ tissues, and the analogous lmaging agents labeled with mr image enhancers, as disclosed hereinabove. These will be packaged separately or together, depending upon whether they are to be in;ected simultaneously or separately, or whether or not they are labeled at the site of administration or at a remote location.

- 2~ -1 3 2 3 3~ 3 The reagents are conveniently provided in kit form, adapted for-use ln the method of the invention.
Kits will normally contain separate sealed sterile vials of in~ectable solutions of labeled reagents, or lyophilyzed antibodies/fragments or antibody/fragment con~ugates and vials of suitable conventional ln~ection vehicles with which they will be mixed Just prior to administration.
Kits may also include reagents ~or labeling antibodies, e.g., Chloramine-T (for I-131 or I-123 labeling), SnCl2 (for Tc-99m labeling using pertechnetate from a commercial generator), short columns for sizing and/or purification of reagents, and other conventional accessory materials.

Without further elaboration, it is believed that one skilled ln the art can, using the preceding description, utilize the present invention to its fullest extent. The following preferred specific embodiments are, therefore, to be construed as merely illustrative, and not limitative of the remainder of the disclosure in any way whatsoever. In the following examples, all temperatures are set forth uncorrected in degrees Celsius; unless otherwise indicated, all parts and percentages are by weight.

Preparation of lym~hoscinti~ra~hic rea~ents.
a) Ga-67-labeled T101 anti-lymphocYte monoclonal antibod~
A sample of T101 anti-lymphocyte murine monoclonal antibody, as reported by Royston et al., loc. cit., is labeled with Ga-67 by the method of Hnatowich et al., Science, 220, 613(1983), to form the con~ugate with a diethylenetriaminepentaacetate (DTPA) gallium(III) chelate, containing an average of 4 Ga atoms per antibody molecule, and retaining at least 70~ of its lnitial immunoreactivity. A solution of the antibody in PBS, pH 7.4, is added to a 50-fold molar excess of solid DTPA dianhydride, and agitated for 5 minutes. Free DTPA is removed by gel filtration on Sephadex G 50. About 1 mCi Ga-67 citrate is added per mg of antibody-DTPA con~ugate, and incubated for 20 minutes, and unbound Ga-67 is then removed, e.g., by gel filtration on Sephadex G 50. The resultant Ga-67-DTPA-T101 has a specific activity of about 0.5 -1.5 mCi/mg.

1323~3 b) I-131-labeled T101 antl-lymphocyte monoclonal antibody A sample of the T101 monoclonal antibody used in Example 1 is labeled with I-131 according to the procedure of Example 1(f) of U.S. Patent 4,348,376, using Chloramine-T and replacing the anti-CEA antibody used in the referenced procedure with an equal weight of T101 antibody, but reducing the amounts of reagents to lower the iodine content. The r~sultant I-131-T101 has an average of 1 atom of iodine per antibody molecule and a specific activit~ of about 12 mCi/mg.
c) In-111-labeled anti-HT~V-1 monoclonal antibody A sample of murine monoclonal anti-HTLV-1 antibody is labeled with In-111 according to the procedure of part (a) hereof, except that In-111 oxinate is used instead of Ga-67 citrate, to form the conjugate with a DTPA indium(III) chelate, containing an average of 3 In atoms per antibody molecule, and retaining at least 70~ of its initial immunoreac-tivity. The resultant In-111-DTPA-anti-HTLV-1 has a specific activity of about 0.5 - 1.5 mCi/mg.
d) In-111-labeled anti-~rostatic_acid phosphatase F(ab')2 A sample of anti-prostatic acid phosphatase (PAP) F(ab')2, prepared by the method disclosed in U.S.
Patent 4,331,647, and described in Goldenberg et al., J.
Am. Med Assn., 250, 630(1983), is labeled with In-111, using the procedure of part (c) hereof. The resultant In-111-DTPA-chelate con~ugate contains an average of 3 In atoms per antibody fragment, and retains at 1east 60% of its initial immunoreactivity. It has a specific activity of about 2 mCi/mg.
e) I-123-labeled anti-CEA monoclonal antibody A sample of the NP-2 monoclonal antibody which specifically binds to carcinoembryonic antigen (CEA), disclosed in U.S. Patent 4,818,709 is labeled with I-123 according to the procedure of Example 1(f) of U.S.
Patent 4,348,376, using Chloramine-T and replacing the I-131 used in the referenced procedure with an equal weight of I-123, but reducing the amounts of reagents to lower the iodine content. The resultant I-123-anti-CEA IgG has an average of 1 atom of iodine per antibody molecule and a specific activity of about 12 mCi/mg.

- 2~ -1323~3 Preparation of in~ectabl compositions Sterile, pyrogen-free solutions are prepared as shown.
a) A sterile solution containing, per ml:
1) 10 m~ Human Serum Albumln (~SA) (1~, USP, Parke-Davls) 2) 0.01 M phosphate buffer, pH 7.5 (Bloware) 3) 0.9~ NaCl 4) 1.5 mg Ga-67-DTPA-T101 antlbody prepared accordlng to Example 1a.
b) A sterlle solution according to Example 2a, except that 250 ug of the I-131-labeled antlbody according to Example 1b is present instead of the Ga-labeled antibody.
c) A sterile solution according to Example 2a, except that 1.5 mg of the In-111-labeled antibody according to Example 1c ls present instead of the Ga-labeled antibody.
d) A sterile solution according to Example 2a, except that 1.5 mg of the In-111-labeled antibody according to Example 1d is present instead of the Ga-labeled antibody.
e) A sterile solutlon accordlng to Example 2a, except that 250 ug of the I-123-labeled antibody according to Example 1e is present instead of the Ga-labeled antlbody.

EXAMP~E 3 Preparatlon of Rea~ents for NMR ~ymPhography a) PreParation of Gd-labeled anti-CEA
A sample of murine monoclonal antibod~ to carcinembryonic antigen (CEA), prepared according to Example 2 of U.S. Patent 4,348,376 or according to Examples 6 and 7 of Canadian Patent Application Serial No. 445.774, is labeled with a p-isothiocyanatobenzoyl-capped oligothiourea containing 320 Gd(III)- DTPA chelate groups prepared according to Example II of copending Canadian Application Serial 510,508, 1323~63 to put an average of 5 oligothiourea chains on the antibody, without loss of more than 30% immunore-activ-ity and without significant aggregation of the antibody con~ugate. The resultant con~ugate carries an average of 320 gadolinium ions thereon. The reaction is effected in 0.1 M aqueous Na2CO3/
NaHCO3 buffer, at pH 8.5, at room temperature, with at least a 50-fold excess of the polymer, and an antibody concentration of about 10 mg/ml.

The con~ugate is purified by gel filtration on a column of allyl dextran cross-linked wlth N,N'-methyl-ene blsacrylamlde, e.g., Sephacryl S-200 (Pharmacia Fine Chemicals, Piscatoway, N.J.).
b ~ aration of Gd(III)-label d T101 monoclonal antlbod~
A sample of T101 murlne monoclonal antibody is labeled with about 320 Gd ions by the procedure of part (a) hereof, except that the antibody is the T101 antibody instead of monoclonal anti-CEA IgG. The resultant con~ugate is isolated by an analogous procedure to the foregoing part of this Example.
C2 PreParation of Cu ~ colloid CuS sulfur colloid is prepared by bubbling H2S
into an acidified solution of CuCl2, in the presence of oxygen and gelatin, and isolating the resultant colloid.

Preparation of in~ectable mri compositions Sterile, pyrogen-free solutions are prepared as shown.
a) A sterile solution containing, per ml:
1) 10 mg Human Serum Albumin (HSA) (1~, USP, Parke-Davis) 2) 0.01 M phosphate buffer, pH 7.5 (Bioware) 3) 0.9~ NaCl 4) 1.5 mg Gd-labeled anti-CEA IgG prepared according to Example 3a.

1323~63 b) A sterile solution accordlng to Example 4a, except that 1.5 mg of the Gd-labeled T101 according to Example 4b is present instead of the Gd-labeled anti-C~A.
., EXAMP~E 5 L~phoscintigraphy A male with proven prostatic carclnoma is belng evaluated for ileopelvic lymph node spread. He is positioned supine on the pelvlc examlnatlon table in the lithotomy position. Two in~ections (approximately 2 mCi each) of Tc-99m-ASC (as supplied by Cadema Medical, Inc., Westtown, N.Y. 10998) and of 111-In-labeled F(ab')2 against prostatic acld phosphatase (PAP) prepared according to Example 2(d) herein, are made just lateral to the anal margin, at 3 and 9 o'clock. The needle is held parallel to the tabletop and is inserted to its full length of l l/2 inches (22-gauge) lnto the ischlorectal fossa. Wlthln each ln~ectlon ls contalned 1 mCi each of the 99m~Tc and the 111-In preparatlon, whlch are mlxed ln the syrlnge for simultaneous application. (It may be preferred to first ln~ect the antlbody fragment and then, 1-2 hrs. later, the antlmony sulfide colloid.) About 3 hrs after the in~ections, the patient is imaged with a medium-resolution collimator, collectlng about 100,000 to 200,000 counts per vlew, followed by repeated imaging.at 8 and 24 hours. Images are taken ln the anterlor, posterlor, and lateral pro~ections.
The images made by the two isotopes with different energies are then computer-subtracted according to the method described by De~and et al., Cancer Res., 40, 3046(1980), whereby the lymph-node image of the Tc-99m-ASC is subtracted, plxel-by-plxel, from that of the In-111-labeled antl-PAP antlbody fragment. At 3 hrs after lnjection of the preparations, and agaln at 8 hrs., the rlght obturator and internal illac nodes are vlsuallzed as havlng abnormal radloactlvlty following perianal in~ection. Images of the 99m-Tc collold alone or of the 111-In-antibody alone were equivocal for lymph node involvement, indicating the superiority of the combined (double isotope and agent) approach.

1323~63 . ., ~XAMPL~ G
Lymphosclnti~raphy A patient with carclnoma of her right breast receives in;ections of approximately 0.25 mCi 131-I-labeled T101 monoclonal antibody, according to Example 2(b), subcutaneously in the web of the fingers of both hands (totalling 0.7 to 1.5 mCi 131-I). The patient also receives, in the same in~ections, 1.5 mCi 123-I-labeled monoclonal anti-CEA antibody NP-2, according to Example 2(e), (totalling 4.2 to 9.0 mCi 123-I). Be~ore administration of the labeled antibodies, the patient is skin-tested for allergic reaction to mouse IgG, and also receives Lugol's iodine to minimize radloiodine concentration by the thyroid gland. Immediately following the subcutaneous ln;ection, the areas are massaged for several minutes, and the patient is asked to exercise her fingers.
By means of a gamma camera, images are obtained at frequent intervals, starting at 2 hrs. after inJection and ending at 36 hrs. The data are stored in a laboratory computer and the images generated on a color display system. The 131-I images are then subtracted, pixel-by-pixel, from the 123-I images by computer processing. The metastatic foci in the ipsilateral axillary lymph node appear as early as 4 hrs., and more clearly at 6 hrs., after in~ection as a discrete focus of increased radioactivity, while the contralateral axillary nodes are negative. Two weeks later, the same procedure is repeated and the results confirmed, except that this tlme. 111 -In is used to label the T101 monoclonal antlbody.
Another study wlth 99m-Tc sulflde collold injected simultaneously with 111-In-labeled anti-CEA
monoclonal antibody, effected as above but with the appropriate doses for these radlonuclides and agents, is repeated a month later and the right axillary lymph node involvement is seen again.

.
L~mphoscintl~ra~h~
A 24 year old male presents with left side inguinal node enlargement and has a constellation of symptoms and history suggestive of AIDS. 67-Ga-- 29 - ~

1323~3 labeled T101 monoclonal antibody, according to Example 2(a), is injected in divided doses into the web of the toes of both feet, with a total dose of 4 mCi, At the same time and in the same inJections, an equal dose of 111-In-labeled monoclonal antibody against HTLV-1, according to Example 2(c), is in~ected. Beginning at 2 hrs post-in;ection and continuing at intervals of 2 hrs up to a total of 6 hrs, and then again at 24 hrs., the patient's inguinal region and pelvis is imaged with a medium-energy collimator and`using the subtraction method described above to subtract the 67-Ga images from the 111-In images. At 4 hrs, and then improving by 6 hrs., a positive image of the left inguinal node is noted while the right inguinal node is vlrtually negative.

Organ Scintigra~hy Hybridoma-monoclonal antibodies are made in the mouse to the Langerhans cells of the endocrine pancreas, derived from a human autopsy speclmen shortly after death. The monoclonals reaetive against the antigen epitopes showing relatively high speeificity for Langerhans cells of the pancreas, as demonstrated, e.g., by immunohistology, are labeled with a gamma-emitting lsotope, such as with I-131, and ln~ected, e.g., 0.15 mg monoclonal agalnst endocrine pancreas antigen, labeled uslng Chloramine-T with I-131, at a dose of 1.0 mCi, in~ected l.v. ln a 3-month old male suspeeted of havlng pathology of the endoerine panereas. External gamma-eamera imaging is performed at 24, 48, 72, and 96 hours after in~eetlon, without subtraetion. In this speeifie ease, deereased to almost absent aecretlon of I-131 radloaetivity in the pancreas is suggestlve of endocrlne pancreas pathology in an lnfant presenting with pancreas hormone deficiency shortl~ after birth.

-- ~0 --,.
.
. The preceding examples can be repeated with .. similar success by substituting the generically or specificall~ described reactants and/or operating conditions of this invention for those used in the preceding examples.
From the foregoing description, one skilled in the art can easil~ ascertain the essential charac-teristics of this invention and, wit~hout departing from the spirit and scope thereof, can make various :
changes and modifications of the invention to adapt it to various usages and conditions.

Claims

1. A lymphographic imaging method for imaging of a tumor or infectious lesion or a localized product thereof in a mammalian lymphatic structure, comprising the steps of:
(a) parenterally injecting a mammalian subject, at a locus and by a route providing access to said lymphatic structure, with an amount of a gross lympho-scintigraphic imaging agent or lymphomagnetic resonance image enhancing agent sufficient to permit a scintigraphic image or an enhanced magnetic resonance image of said structure to be effected; and (b) obtaining a gross scintigraphic image or a gross enhanced magnetic resonance image of said structure, at a time after injection of said agent sufficient for said agent to diffusely accrete in said structure;
wherein said gross imaging agent comprises an antibody/fragment which specifically binds to normal lymphatic cells or tissues, said antibody/fragment being labeled with a radioisotope or a magnetic resonance enhancing agent.

2. A lymphographic imaging method for imaging of a tumor or infectious lesion or a localized product thereof in a mammalian lymphatic structure, comprising the steps of:
(a) parenterally injecting a mammalian subject, at a locus and by a route providing access to said lymphatic structure, with an amount of a gross lymphomagnetic resonance image enhancing agent sufficient to permit an enhanced magnetic resonance image of said structure to be effected; and (b) obtaining a gross enhanced magnetic resonance image of said structure, at a time after injection of said imaging agent sufficient for said agent to diffusely accrete in said structure.

3. The method of claim 2, wherein said gross imaging agent comprises an antibody/fragment which specifically binds to normal lymphatic cells or tissues, said antibody/fragment being labeled with a magnetic resonance enhancing agent.

4. A method of organ imaging in a mammalian subject by scintigraphy or magnetic resonance imaging, comprising the steps of:
(a) parenterally injecting a mammalian subject, at a locus and by a route providing access to an organ of interest, with an antibody or antibody/fragment which specifically binds a marker produced by or associated with said organ, said antibody/fragment being labeled with a radioisotope capable of external detection or with a magnetic resonance image enhancing agent, the amount of the labeled antibody/fragment being sufficient to permit a scintigraphic image or an enhanced magnetic resonance image of said organ to be obtained; and (b) obtaining a positive scintigraphic image or a positive enhanced magnetic resonance image of said organ, at a time after injection of said agent sufficient for said agent to diffusely accrete in said organ and specifically bind to said marker.

5. A lymphoscintigraphic imaging kit for imaging a mammalian lymphatic structure, comprising:
(a) at least one gross lymphoscintigraphic imaging agent, which comprises an antibody/fragment which specifically binds to normal lymphatic cells or tissues, said antibody/fragment being labeled with a radioisotope label; and (b) a sterile, pharmaceutically acceptable injectable vehicle for injection of (a).

6. The method of claim 4, wherein saidmarker is an intracellular marker.