CA1338706C - Monoclonal anti-human breast cancer antibodies suitable for imaging breast cancer - Google Patents

Abstract

Hybridomas producing monoclonal antibodies suitable for imaging and diagnosis of human breast tumors and such monoclonal antibodies are claimed. The monoclonals are characterized by breast tumor binding range, breast cancer cell line range, and selectively.
Immunoimaging agents comprising the monoclonal antibody and a detectable label, either directly or indirectly conjugated to the antibody are claimed. Methods for imaging breat tumors using the immunoimaging agents are described and claimed.

Description

338~6 PATENT
Case No. 2212.1 r ~ r AL ANTI-HlMAN BREAST CANCER ANTIBODIES
This invent~on is in the fields of immunology and cancer diagnosis and therapy. More particularly it concerns murine monoclonal anti-human breast cancer antibodies, hybridomas that 5 produce those antibodies, ~mmunochemicals made from those antibodies, and diagnostic and therapeutic methods that use those immunochemicals.
Since the mid-1970s, there have been numerous reports of murine monoclonal antlbodies that interact with human breast cancer associated antigens. In these reported studies, mice were immunized 10 and boosted with human m~lk fat globule proteins, breast cancer cell lines or breast cancer membrane extracts. Immune splenocytes were fused with mouse myeloma cells and hybridomas were selected based on some specificity of the culture media for breast or breast cancer antigens. Taylor-Papadimitriou, J., et al, Int. J. Cancer (1981) 15 28:17-21; Yuan, D., et al, JNCI (1982) 68:719-728; Ciocca, D. R., et al, Cancer Res. (1982) 42:4256-4258. The normal tissue reactivities of these prior antibodies are dlfferent than the normal tissue reactivities of the antibodies of the present invention.
A principal aspect of the invention concerns murine 2 0 monocl onal ant i bod~ es that:
(a) do not bind to blood cells;
(b) have a breast tumor binding range of at least 0.25 or have a breast cancer cell line binding range of greater than or equal to 0.25;
(c) have a selectivity equal to or less than 0.09;
(d) h2ve a G or M isotype, and (e) when conjugated to an ~maging moiety, produce a signal sufficient to image breast cancer tumors.

2 13387~6 Preferred embodiments of ~hese antibodies are those des~gnated 2G3, 9C6, 32A1, 33F8, 35E10, 41B4, 87H7, 106A10, 113F1, 120H7, 14DA7, 200F9, 203E2, 219F3, 245E7, 254H9, 260F9, 266B2, 317GS, 369F10, 387H9, 421E8, 451C3, 452E12, 452F2, 454A12, 454C11, 457D7, 5 520C9, 65OE2, 697B3, 741F8, 759E3, 788G6, and functional equivalents thereof .
The murine x murjne hybridomas that produce the above described antibodies and progeny of those hybridomas are other aspects of the invention.
Another aspect of the ~nvention relates to immunoimag~ng agents that are conjugates of (a) the above described monoclonal antibodies, and (b) a detectable imaging moiety.
Another aspect of the invention concerns methods of imaging 15 breast tumors in a patient in need of such imaging by administering an imaging effective amount of an immunoimaging agent and detecting the immunoimaging agent in the patient with a suitable detecting device.
As used herein, the term "monoclonal antibody" means an antibody composition having a ~ s antibody population. It is 20 not intended to be limited as regards the source of the antibody or the manner in which it is made.
As used herein with regard to the monoclonal antibody-producing hybridomas of the ~nvention the term "progeny" is intended to include all derivatives, ~ssue, and offspring of the parent 25 hybrldoma that produce the monoclonal anti-human breast cancer antibody produced by the parent, regardless of generation or ka ryotyp i c i dent i ty .
As used herein with respect to the exemplified murine monoclonal anti-human breast cancer antibodies, the term "functional 30 equivalent" means a monoclonal antibody that: (a) binds to the same antigen or epitope as an exemplified monoclonal ant~body; (b) has a breast tumor binding range of at least 0.25 or has a breast cancer cell line range of greater than or equal to 0.25; (c) has a ~- 1338706 selectivity equal to or less than 0.09,, (d) has a G or ~1 isotype, and (e) when conjugated to an imaging moiety, produces a signal sufficient to ~mage breast cancer tumors.
As described above, the term "functional equivalent" as used 5 herein includes five criteria. The first of these criteria, binding to the same antigen or ep~tope as an exemplified monoclonal antibody may be demonstrated by experiments which show crossblocking of an exemplified monoclonal antibody by the functionally equivalent monoclonal antibody. Crossblocking occurs as a result of an antibody 10 binding to the same epitope on an antigen as that bound by one of the exemplified antibodies, or as a result of an antibody binding to a different epitope which is so closely situated on the same antigen that binding of an antibody to one epitope blocks the binding of an antibody to the second epitope. Crossblocking thus is one of the 15 criteria by which one can determine that a functionally equivalent monoclonal antibody binds to the same antigen or epitope as an exemplified monoclonal antibody.
So-called "sandwich" assays are another method for determining whether an antibody binds the same antigen or epitope. In 20 these assays, a first monoclonal antibody is bound to a support, for example, the surface of a titre plate well. After treatment to prevent nonspecific binding, a highly solubilized antigen preparation is added to the bound antibody. cuhs~q ~ ly, a second antibody, having a detectable label, for example, a fluorescent dye, is added.
25 If the second antibody binds to the antigen, a dlfferent epitope specificity or multiple copies of the same epitope on the same antigen is indicated. If the second ~ntibody fails to bind. either the same epitope specificity or different antigen specificity is indicated.
The results of both the crossblocking and sandwich assay are further 30 defined by a second series of tests such as immune precipitation or Western blotting to show that the antigen bound by both antibodies has the same mol ecul ar wei ght.

~- 13387~6 Mo nocl onal Ant i body P rodu ct i on The antibody-producing fusion partners that are used to make the hybridomas of this invent~on are generated by immunizing mice with live human breast cancer cells or membrane extracts made therefrom.
5 The mice are inoculated intraperitoneally with an immunogenic amount of the cells or extract and then boosted with similar amounts of the immunogen. Spleens are collected from the immunized mice a few days after the final boost and a cell suspension is prepared therefrom for use in the fusion.
Hybridomas are prepared from the splenocytes and a murine tumor partner us~ng the general somatic cell hybridization technique of Kohler, B. and Milstein, C., Nature (1975) 256:495-497 as modified by Buck, D. W., et al, In Vitro (1982) 18:377_381. Available murine myeloma lines, such as those from the Salk Institute, Cell 15 Distribution Center, San Diego, California, USA, may be used in the hybridization. Basically, the technique involves fusing the tumor cells 2nd splenocytes using a fusogen such as polyethylene glycol.
After the fusion the cells are separated from the fusion medium and grown in a selective growth medium, such as HAT medium, to eliminate 20 unhybridized parent cells. The hybridomas are expanded, if desired, and supernatants are assayed for anti-human breast cancer activity by conventional immunoassay procedures (e.g., radioimmunoassay, enzyme csay, or fluorescence immunoassay~ us~ng the immunizing agent (breast cancer cells or membrane extract) as antlgen. Positive clones 25 are characteri zed further to determi ne whether they meet the criteri a of the antibodies according to the invention.
Hybridomas that produce such antibodies may be grown l n v~tro or in vivo using known procedures. The monoclonal antibodies may be isolated from the culture media or body fluids, as the case may 30 be, by conventional immunoglobulin purification procedures such as 2mmonium sulfate precipitation, gel electrophoresis~ dialysis, chromatography, and ultrafiltration, If desired.

5 133870~
Monoclonal Antibody Selection/Characterkation The important characteristics of the monoclonal antibodies ~re (I) their immunoglobulin cldss, (2) their selectivity for human breast cancer cells, (3) the range of human breast cancer tumor cells 5 to which they bind, (4) the range of human breast tumor frozen sections to which they bind, and (5) their usefulness in making effective anti-human breast cancer immunoimaging agents.
The selectivity and range of a given ant~body is determined by testing it against panels of (1) human breast cancer tumor tissues, 10 (2) human breast cancer cell lines, and (3) normal human tissue or cells of breast or other origin. In selecting the claimed antibodies, approximately 22,000 growing hybridoma cultures were initially screened against the immuniz~ng breast tumor membranes or cell line, a panel of seven normal tissue membranes, a fibroblast cell line and a 15 breast tumor frozen section. Clones that reacted with the neoplastic materials, but not the normal materials, were identified in this initial screen and chosen for isotyplng and additional screening for selectivity and range. The additional screening involved: sixteen normal tissue sections, five normal blood cell types, eleven nonbreast 20 neoplasm sections, twenty-one breast cancer sections and fourteen breast cancer cel 1 1 i nes .
For purposes of this patent application, specificity and selectivity are used interchangeably and are defined as the sum of the number of substructures stained in sixteen normal tissue frozen 25 sections and the number of blood cell types bound, divided by the sum of the total number of substructures bound by any of the monoclonal antibodies in all the tissue on which the monoclonal antibodies were tested and five blood cell types tested.
The term "tumor range" is defined as the number of breast 30 tumor frozen sections stafned divided by the number of breast tumor frozen sections tested. The term breast cancer "cell line range" is defined as the number of breast cancer cell l~nes sta~ned divided by the number of breast cancer cell lines tested. Antibodies were deemed to be appropr~ate for breast cancer immunoimaging purposes if they 6 13387~6 have a selectivity equal to or less~than 0.09 and a breast tumor binding range of equal to or greater than 0.25 or a breast cancer cell line binding range of equal to or greater than 0.25.
Antibodies exhibiting acceptable selectivity and range may 5 be conjugated to various ~maging moieties such as radioisotopes or materials detectable by nuclear magnetic resonance imaging. In some cases a coupling agent, such as a chelating agent may be used to link the imaging agent to the antibody.
Antibodies of five of the thirty-three deposited hybridomas 10 were found to recognize the same 200 K dalton antigen. Antibodies of four of the thirty-three bound to a 230 K dalton intracellular antigen. Three bind to one or more high molecular weight mucins (HllW) and two bound to transferrin receptors in the form of a 97 K dalton antigen. All antigen weights mentioned herein were determined by 15 sodiu~ dodecyl sulfate (SDS) polyacrylamide gel electrophoresis under reducing conditions using procedures known in the art.
Further details of the characterization of these antibodies are provided in the examples below.
Immu nochemi cal s The immunochemical derivatives of the monoclonal antibodies of this invention that are of prime importance are labeled with an imaging moiety such as radioisotopes, radiopaque substances or nuclear magnetic resonance detectable materials. Such immunochemical derivatives, in which the imaging moiety provides a means for identifying immune complexes that invlude the labeled antibody may be used in imaging breast cancer tumors in vivo.
Antibodies that exhibit either a breast cancer tumor binding range of at least 0.25 or a breast cancer cell line binding range of at least 0.25, and that also exhibit a selectivity equal to or less than 0.09 and do not bind to blood cells, were considered selective for breast cancer immunoimaging purposes, and may be conjugated to a detectable imaging moiety. Such ~maging moieties may be directly bound to the monoclonal antibody or may be bound to the monoclonal . , . .. . . . .. ... . . . . .. .. . .... . . . . .. . _ . . . .. . . .. ... . ... . ..

-ant~body by means of a linking or chelating agent. ~erivatives of the monoclonal antibody, labeled wth the imaging moieties, can be made by a variety of methods well known in the art. Such labeled derivatives are also referred to herein as immunoimaging agents.
Radioisotopes of iodine may be used to iodinate monoclonal antibodies using the sol~d phase oxidizing agent ,13,4,6-tetrachloro-3~,6~-diphenylglycouril (sold under the tradename lodo-gen~), or N-chloro-p-toluene sulfonamide (chloramine T).
The term "linkers" used herein is intended to encompass chemical entities which may be bound to the imaging moiety and which also bind to the monoclonal antibody. Appropriate linkers may include those which bind to the monoclonal antibody and chelate radionuclides. Other linkers, such as those which may selectively bind to the carbohydrate carrying regions of the monoclonal antibody, or those that are capable of binding free amino side groups of the protein region of the monoclonal antibody, such as amidinating or imidinating agents, and whlch can be covalently linked to the imaging moiety, are also included in the scope of the term linker as used herei n.
Appropriate linkers will have three characteristics. First, they must be capable of bind~ng the imag~ng moiety which has the desired characteristic to be read for imaging. Secondly, the linker must not s~gnificantly affect the binding selectivity of the monoclonal antibody or substantially diminish its affinity for the antigen to be bound. Lastly, the linker must form a stable bond with the imaging mo~ety and the monoclonal antibody so that the imaging moiety and antibody will not be separated from one another.
The particular linker and imaging moiety used to make the ~mmunoimaging agents of the present invention will vary from ant7body to antibody depending on the effect that a particular imaging moiety or imaging moiety and linker may have upon the binding characteristics of the monoclonal ant~body for the target antigen. Thus, while one monoclonal antibody may be iodinated with a radioisotope of iodine at tyrosine resldues w~thin the monoclonal antibody without significantly ~ . , . . . ... .. .. .. . .. . . . . . _ . . . . . . . .. .. .

~ 1338706 affecting affinity or selectiv~ty of the monoclona1 antibody, the same treatment of a second monoclonal ant~body according to the ~nvention may significantly diminish the affinity or binding specificity of another monoclonal antibody. A different label or linker, for 5 example, one that binds to the antibody at a different amino acid residue, may be used without affecting selectivity or affinity in the second antibody. Thus, iodine radioisotopes can be linked to the second antibody, for example, using the method of Wood, F. T., et al.
Analy. Biochem. 69:339 (1975) and the linker methyl-p-hydroxybenz-10 imidate or the method of Bolton-Hunter, Bolton, A. E. and Hunter, W.
M., Biochem. J. 133:529-539 (1973) and the linker N-succinimidyl-3-(4-hydroxyphenyl) propionate. A chelating agent such as diethylinetriaminepentaacetic acid anhydride which binds to lysine residues of the antibody, or ethylenetriaminetetraacetic acid may be 15 used to label the antibody with 111-Indium (111-ln), and could also be employed as an alternative means for linking the antibody to the ~maging moiety. See for example, Goodwin, et al., "Chelate Conjugates of Monoclonal Antibodies for Imaging Lymphoid Structures in the Mouse', J. Nucl. Med. 26(5):493-502 (1985) and Meares et al., 20 "Conjugation of Antibodies With Bifunctional Chelating Agents Bearing Isothiocyanate or Bromoacetamide Groups and Subsequent Additon of Metal Ions", Analy. Biochem. 142:68-78 (1984).
Various moieties suitable for imaging are known. For example, monoclonal antibodies have been radiolabeled with a number of 25 radionuclides suitable for imaging, including 131-iodine (1-131) and 1-123. Levin et al., "Localization of I-131 Labeled Tumor Bearing Balblc Mouse", J. Nuclear Medicine, 21:570-572 (1980); Farrands et al., "Rad~oimmunodetection of Human Colorectal Cancers by an Anti-Tumor Monoclonal Antlbody", Lancet 397-399 (1982); Zimmer et al., 30 URadioimmunoimaging of Human Small Cell Lung Carcinoma With I-131 Tumor Specifi~ Monoclonal Antibody", Hybridoma, 4(1):1-11 (1985). The direct label~ng of the monoclonal antibody with radioisotopes of iodine can be carried out according to the methods described in Contreras et al, Methods in Enzymology (1973) 97:277. Technetium-99 35 has been used as an imaging moiety; Khaw et al., "Monoclonal Antibody ... . . .. . . . . . . . . .. .. ....

to Cardiac Myosin: Imaging of Experi Rental Myocardial Infraction", Hybridoma 3:11-23 (1984). 111-In has been applied as a label for antibodies, Krejack et al., "Covalent Attachment of Chelating Groups to Macromolecules", Biochem. Biophys. Res. Comm., 77:581-585 (1977);
5 Hnatowich et al., "Radioactive Labellng of Antibody A S~mple and Efficient Method", Science, 220:613-615 (1983) and Schienberg, D. A.
et al., "Tumor Imaging With Radio2ctive Metal Chelates Conjugated to Monoclonal Antibodies", Science, 215:1511-1513 (1982).
In order to initially assess the suitability of the antibody 10 as one appropriate for imaging, the antibody may be labeled with a moiety that is directly detectable such as fluorochromes, as well as moieties, such as enzymes, that must be reacted or derivatized to be detected. Examples of such labels are fluorescein and its derivatives, rhodamine and its derivatives, dansyl groups, 15 umbelliferone, luciferin, 2,3-dihydrophthalazinediones, horseradish peroxidase, alkaline phosphatase, lysozyme, and glucose-6-phosphate dehydrogenase. The antibodies may be tagged with such labels by known methods. For instance, coupling agents such as aldehydes, carbodiimides, dimaleimide, imidates, succinlmldes, bis-diazotized 20 benzidine and the like may be used to tag the antibodies with the above-described fluorescent, chemiluminescent, and enzyme labels.
The antibodies and labeled antibody may be used in a variety of immunoimaging or immunoassay procedures to detect the presence of breast cancer in a patient or monitor the status of such cancer in a 25 patient already diagnosed to have it. When used for ~n vivo immunoimaging to detect the presence of a tumor, its location and dissemination 1n a patient's body and the progress of therapy to ameliorate the tumor load, the monoclonal antibody labeled with an imaging moiety will be administered parenterally, preferably 30 intravenously or subcutaneously in an amount sufficient to accumulate at the tumor site and be detected by the detecting means of choice.
Typlcally the monoclonal antibody labeled with an imaging moiety will be administered with a suitable pharmaceutically acceptable carrier of the type well known to those skilled in the art. Such carriers do not 35 affect the patient. The amount of monoclonal antibody to be . _ _ . _ _ . . .

administered will depend upon the amount of detectable imaging moiety attached to the monoclonal antibody and the residual binding efficiency of the monoclonal antibody after labeling wlth the imaging moi ety .
The residual binding efficiency of the monoclonal antibody labeled wlth the imaging moiety Is determined in vitro using a tumor cell binding assay. Generally, radioimmunoreactivity, which measures the residual binding efficiency of a radiolsotope-labeled monoclonal antibody is determined by comparing specific binding of the radioisotope-labeled monoclonal to a fixed tlssue culture of a known immunoreactive tumor cell line such as SKBR-3, MCF-7, and MX-l with non-specific binding to a fixed cell line which does not specifically bind the monoclonal.
The optimal radioimmunoreactivity of the labeled monoclonal is determined in this system by varying the concentration of the imaging agent available for binding to the monoclonal antibody while keeping the concentration of the monoclonal antibody constant. The labeled monoclonals are then tested in the flxed cell immunoassay des~ribed above by adding the labeled monoclonal to the fixed cells at conditions of antigen excess. The labeled monoclonal giving the highest detectable binding is determined and can be used initially for in vivo radioimmunoimaging.
When an in vitro ~mmunoassay Is used to monltor the status of a cancer patient, a quantitative i~ ~o~csay procedure must be used. In such monitoring, assays are carried out periodically and the results compared to determ~ne whether the patient's tumor burden has ~ncreased or decreased. Common assay techniques that may be used include direct and indlrect assays. Direct assays involve Incubating a tissue sample or cells from the patlent w~th a labeled antibody. If the sample includes breast cancer cells, the labeled ~ntibody will - bind to those cells. After washlng the tissue or cells to remove unbound labeled antibody, the tissue sample is read for the presence of labeled Immune complexes. In Indirect assays the tissue or cell sample is Incubated with unlabeled monoclonal antibody. The sample is , , _ . _ .. . . , . , . , ~ .

~ 1338706 then treated with a labeled antibody a~ainst the monoclonal antibody (e.g., a labeled antimurine antibody), washed, and read for the presence of labeled ternary comp1exes.
For in vitro diagnostic use the antibodies will typically be 5 distributed in kit form. These kits will typically comprise: the antibody in labeled or unlabeled form in suitable containers, reagents for the incubations and wash1ngs, a labeled antimurine antibody if the kit is for an indirect assay, and substrates or derivatizing agents depending on the nature of the label. For in vivo imaging use the 10 antibody will also be distributed in kit form and will typically comprise the same types of cr ~ s as mentioned above. The antibody may be supplied derivatized with an agent already bound to or chelated with the radioisotope to be used, or the monoclonal may be supplied derivatized with the binding or chelating agent only, and the 15 radioisotope to be used may be supplied separately. The radioisotope to be used can be added just prior to use so that an optimal radioactivity level for imaging can be achieved at the time of administration of the radioimmunoimaging agent to the patient. Human breast cancer antigen controls and instructions may also be included 20 if appropriate to the test.
The following examples provide a detailed description of the preparation, characterization, and use of representative monoclonal antibodies of this invention. These examples are not intended to limit the invention in any manner.
25 Immunization Fresh postsurgical human breast cancer tissue and a variety of normal tissues were used to prepare membrane extracts by h omogen i zat i on and di scont~ nuous suc rose g radi ent cent ri f ugati on .Human breast cancer cell lines were obtained from the Breast Rncer ~0 Task Force, the American Type Culture Collection (ATCC), and from Dr.
Jorgen Fogh at Memorial Sloan Kettering. The cells were maintained and passaged as rec~ ' by the Breast Cancer Task Force, the ATCC
and Dr. Fogh. For ~mmunizations, either membrane extract containing 12 1338~06 100 1~9 of protein (Lowry assay) or ten million liYe breast c2ncer cells were ~noculated intra-peritoneally into five week old Balb/c m~ce. The mice were boosted identic211y twice 2t monthly interv21s.
Three d2ys 2fter the 12st boost, the spleens were removed for cell fusion-Hybridom2 Methods Somatic cell hybrids were prep2red by the method of Buck, D.
W., et 21, supr2, using the murine myeloma line Sp-2/0/Ag14. All hybrodima cell lines were cloned by limiting dilution. Half of the fusions employed splenocytes from mice immunized with breast c2ncer membr2ne extr2cts 2nd h21f used splenocytes from mice immunized with live bre2st c2ncer cell lines. Eighty-three thousand four hundred twenty-four wells were generated from those fusions, of which 22,459 exhibited hybridoma growth.
Screening Methods Hybridoma supern2tant w2s 2ss2yed for re2ctive 2ntibody in either 2 solid ph2se enzyme-linked immunosorbent 2ssay (ELISA) with the immunizing breast cancer membr2ne extr2ct or an indirect 1mmunofluorescence 2ss2y with the immunizing bre2st c2ncer cel 1 line. For the solid phase membr2ne ELIS~, 40 ~1 of 0.1 mg/ml bre2st c2ncer membr2ne protein were pl2ced in polyvinyl chloride (PVC) microtiter wells for 12 hours at 4C. The extr2ct w2s 2spir2ted and the wells w2shed with phosphate buffered saline (PBS) contain~ng lX
bov~ne serum albumln (BSA). The wells were then incub2ted with 45 1l1 of 2 l:IO dilution of hybridoma supern2t2nt. The diluent was medi2 w~th 25 mM of a buffer, 10% bovine serum, 2nd 0.1X sodium 2zide.
After 30 m~nutes 2t room temper2ture, the wel l s were 2g2i n w2shed 2nd incub2ted 45 minutes 2t 37C w~th 2 1:200 dilution of perox~dase conjugated go2t 2nti-mouse IgG. The diluent W25 PBS. The wells were then w2shed with PBS 2nd reacted with 200 ~1 of 1,2-2zino-di(3-ethylbenzthi2zoline sulphonic 2cid) in 0.1 M sodium citrate buffer pH

4.2 for 30 minutes at room~emper2ture. Optic21 denslty w2s measured ~,-j at 405 nm on a MicroElisa Reader. For each experiment a positive ~ l~d~ ItCL,f~
~ , 13 1~8706 control, anti-beta 2 microglobulin at 5~~g/ml, was reacted with normal human kidney membrane. This gave an optical density of 1.0 i 0.1 (standard deviation). The background was O $ 0.1 optical density units (O.D.) using media without mouse monoclonal antibody. Well s 5 that gave a reaction on the breast cancer membrane extract of greater than 0.7 O.D. were saved.
For the indirect immunofluorescence cell line assay 100,000 breast cancer cells of the immunizing cell line were placed overnight with appropriate media in each chamber of a set of eight chambered lO slides. Similarly, 100,000 fibroblast cells from cell line CC95 were incubated overnight in chambered slide wells. The cells were washed with PBS containing lX BSA. The wells, both breast cancer and fibroblast, were incubated for 30 minutes at 4C with 1:10 dilutions of hybridoma supernatant. The cells were again washed and incubated 15 30 minutes at 4C with a 1:50 dilution of fluorescein isothiocyanate (FlTC)-conjugated goat F(ab' )2 anti-mouse Ig. The cells were washed three times, fixed in 1.5~ formaldehyde in PBS for five minutes, chambers removed and rinsed in PBS. The slides were then mounted in a composition containing polyvinyl alcohol, glycerol, buffers and a 20 preservative and examined with a fluorescence microscope. Hybridoma wells showing strong fluorescent binding to the breast cancer cells but no fluorescent binding to fibroblasts were saved. F1ve thousand one hundred fifty-six hybridoma wells revealed breast cancer reactivity in the initial screen.
Supernatants from the 5156 positive wells were then tested in solid phase ELISA w~th seven normal tissue membrane extracts (liver, lung, colon, stomach, kidney, tonsil, and spleen). Any well supernatant giving an ELISA O.D. greater than 0.3 was discarded. One thousand one hundred one of the supernatants were found to be unreactive with the normal tissue extracts.
- The 1101 hybridoma supernatants were tested on frozen sectlons of human breast carcinoma tissues. Six micron sections were attached to slides, fixed 10 minutes in acetone at 4C, dried 10 minutes at room temperature, washed wlth PBS, blocked with horse serum 14 133870~
and incubated 20 minutes at room ~emperature with 100 ~l neat hybridoma supernatant. The slides were washed with P~S, and finally incubated 20 minutes at 37C with a 1:50 dilution of peroxidase conjugated rabbit anti-mouse Ig, washed again with P8S, and finally 5 incubated 7.5 minutes at 37C with O.S mg/ml diaminobenzidine in û.OS
M Tris buffer pH 7.2 containing 0.01X hydrogen peroxide. The slides were stained with hematoxylin, dehydrated and mounted in a medium containing 35.9X methyl/n-butylmethacrylate copolymer, 7.1X butyl benzyl phthalate, and 0.3X 2,6-ditertbutyl-p-cresol. One hundred 10 twenty-four wells yielded breast cancer selective binding and were c l oned .
Purification and Class Determination Immunoglobulin class and subclass of the monoclonal breast cancer selective antibodles were determined by an immunodot assay 15 essentially the same as that described in McDougal et al. J. Immunol.
Meth. 63:281-290 (1983). Antibodies were also internally labeled by growing 2-3 x 106 hybridoma cells for four hours in methionine-free medium containing 0.2 IICi 35S methionine. 35S-labeled antibodies were immunoprecipitated with fixed staphylococcus A cells, or with fixed 20 staphylococcus A cells precoated with rabbit anti-mouse lmmunoglobulin, and the immunoprecipitates were analyzed by SDS-PAGE
to determine antibody light and heavy chain mobility, lack of extra chai ns, and the abi l i ty of each anti body to bi nd staphyl ococcal protei n A.
The antibodies were expanded in vivo. Balb/c or F1 (C57B/6 x Balb/c) mice were primed with 0.5 ml pristane intraperitoneally (ip) and after 10-14 days inoculated with one million log phase hybridoma cells in PBS. Ascites fluid was stored at -70C and thawed and filtered through a 0.8 micron filter unit before further purification.
Some IgG antibodies that bound staphylococcal protein A were purified by affinity chromatography on prote~n A-chromatographic resin containing e~ther agarose, dextran and/or acrylamide w~th pH step gradient elution. IgG ant~bodies that did not bind protein A were precipitated by addition of ammonium surfate to 40~ saturation at 0C.
or by binding to DEAE or Affigel`' (Biorad, Richmond, Californi a).
Alternatively, Ig6 ant~bodies are purified by chromatography using a Sephacryl S-200 column, followed by DEAE cellulose as described. The 5 precipitates were redissolved in PBS, dialysed to 20 nM Tris pH 7.2 and chromatographed on a 1.6 x 50 cm column of diethylaminoethyl cellulose (DEAE) eluting w~th a l.S liter 0-600 nM NaCl gradient at 4C at a flow rate of 1 ml/min. In each case, column fractions were monitored by SDS-PAGE and the purest antlbody fractions were pooled, lO concentrated to 1-3 mg/ml, di alysed to PBS/0.02X NaN3, and stored at 4C .
IgM antibodies were pur~fied by gel filtration material on a 2.6 x 40 cm column of Sephacryl S-300 or other gel filtration or resin containing agarose, dextr3n and/or acrylamide, eluting with PBS/0.01%
15 sodium azide at room temperature at a flow rate of 1 ml/min.
SelectiYity Determination In order to evaluate their selectivity for breast cancer, the purified antibodies were tested by immunoperoxidase section staining on sections of sixteen normal tissues, and by 20 immunofluorescent cell sorting on five blood cell types.
Immunoperoxldase staining was performed as above except that known dilutions of purified antlbodies in P3S in the range of 1-40 Ilg/ml were used instead of hybridoma supernatants. The pure antibodies were first titrated to find the minimal concentration giving strong 25 immunoperoxidase stainlng on breast cancer sections and then used at the concentration for the normal tissue tests. Peripheral bood cells (platelets, lymphocytes, red blood cells, granulocytes, and monocytes) were prepared by centrifugation using a medium which separates monocytes from pol~, "~ lear leucocytes. The cells were reacted 30 with antibody at the optimal concentration determined above for 30 minutes at 4C, washed. reacted with a 1:50 dilution of fluorescein ~sothiocyanate-conjugated goat anti-mouse Ig for 30 minutes at 4C, washed again and examined in a cell sorter. The wash buffer and diluents were PBS w1th lX gelatin and 0.02X sodium azide. The cell sorter was equipped with a 76 micron n~zzle and a one watt argon ion laser at 488 nm. An 80 mm confocal lens was used on the optical rail assembly for focusing. Other filters used were a 515 nm interference f~lter and a 515 nm absorbance filter (for scattered laser light) and 5 a neutral density 1.5 filter for forward angle light scatter. Contour plots of log fluorescein fluorescence versus forward angle light scatter were used for sample analysis. No blood cell types showed detectable binding.
The binding behaviors of the claimed antibodies are reported 10 in Table I below. The following abbreviations are used to denote structures bound by the ant1bodies: Ac, acini; G, glands; T, tubules;
D, ducts; L, lumen; W, sweat glands; E, epithelium; S, sebaceous glands; Gr, granulocytes; Mk, megakaryocytes; M, macrophage; Ly, lymphocytes; Bl, Basal layer; Fe, focal epithelium; A, alveolar lining 15 cells; B, Bowman's capsule; Mu, muscle; and I, islets; H, hair follicles; U, glomeruli; and V, vessels/endothelial.

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' o o C~ o o 2--' o o o o ~ _ -- o o _ o --o o o o o -- o o o o o o ~, ~ o o o o o _ o o o o o o o o o o o _. o o o o o o _ o o o ~ o o o o r! ~ m :~ ~ O ~ -- o o O O o ~ ~ ~ ~ ~ o c~, _ -- ~ o o ~ O o o O _ O -- ~ ~ O O ~
~ _ O _ O _ O O _ O O O O ~ O _ _ O o o O o o c~ O o _ O o o _ o o o o o ~
D ~ Ol ~ O~ ~ O -- -- Ol -- O O ~ ~ O C`~ ~ ~ O O O ~ O O O O --' O O O O O O O
C _ ¢o_ooO-~o~ ¢~o-~ooOo OO~OOOO ~ ~D
m --00-- ~ ~ ~-- ~ w ~ _ ~ ~ O 0 ~0 ~ ~) ~ o ~ r~ 'd' O ~ S `O ~ o o~ ~ ~ O ~ ~

~ 13~87~

Breast Cancer Tumor Binding Range Determlnation In order to determine how wide a range of breast cancers might be recognized by each ~nt~body, the breast cancer selective ~nt~bodies were tested by ' ~ r~dase st~ining on frozen section 5 of 27 different breast tumors. The breast cancers used for section st~ining were all ~nfiltrating ~ntr~ductal carclnomas, so no correlat~on of ~ntibody bindlng with histo~ogic type of breast cancer could be made. In addition, no correlat~on between antibody binding ~nd the nodal status or estrogen receptor status was found for the 10 twelve tumors for which donor information was available. Antibodies reacted equally well with metastatic and primary breast tumors. The results of these tests for the claimed antibodies are reported in Table 2 below.

1338~06 o o ~ ~ o ~ ~ ~ ~1 ~ o o o ~ o o o o o ~ o o o o o c~ o ~ ~ ~ rl ~ -- -- -- ~ ~ ~ o o o o -- -- o ~ c~ o o ~ ~ _ o o cY o ~ ~ o ~ o - - - o - - - - c~ o o c~
o o C ~ o ~ ~ o ~ o ~, o o o o o o o ~ o o o o o o C`l ~ o o C`l o o C~ ~ o -- o o C l o C: ~ ~ C l o C~ ~ o o o C~ o o C ~ o ~ o o o o o o o o o o C`~ o ~ o ~ o ~ C`l ~ ~ ~ o 0-O~o~-o-~ 0~ 00-~O~ooooooo_~oo~
~ o o C~ C l o o C l o ~ o C l o o o o ~ o o o ~ ~ -- o c~l o _ ~ o o ~`I o t~l ~I o _ o _ o _ r O _ ~I _ O _ _ _ _ C~l o ~l~z ~
m ttl ~0OOOOOOO~O -~ ~0OO_~OOO_OOO~OOO
_ O O O _ ~ O O ~ O O ~ ~ _ O O _ O O O O _ O ~ O ~ O _ ~ ~ O
O O ~ ~ ~ ~-- --. O O C`~ ~ O ~
~' ~ ~ O O ~ O _ -- O O O ~ -- ~ -- -- _ O O O O _ ~' ~ ~ O O O O ~ ~ O O ~ O ~ ~ O O -- O O _ _ O O ~ _ O O _ O O O O --_ O ~ ~ O ~ O ~ _ O ~ ~ ~ ~ --_ ~ o o o _ ~ _ .-- o _ o rl ~ O O O ~ -- -- _ O O O O O O ~ c~l O O C~
m ~ 0~OO C`le`l 0--C`IO~O~O__~O_OOOC`~
~ ~o ¢ ~ ~~ P: ~C ¢ ~" ~ ¢ ~ $ C~ ~ m ~ ~ ~
X _ _ _ ,_ _ _ _ _ _ _ _ _ U~
m ~o ~1 ~
V
m m ~
CC
CC - ~ O - ~ ~ O O ~
_ O ~ O O ~ O O O
m O 0-O.-OO~-O-~
O-.O-O~O~OO~~O~C`100 ¢
O O O ~ ~ ~ O ~ C~ ~ ~ ~ ~ ~ O C~ O ~ ~ ~ o o -- -- o ~ rl o o c~
1 ~ ~ o o c~ ~ ~ ~ o o o ~ ~ r~ -- o c~l o o o _ o o -- o o o ~ o o c~
~ ~ L~ m ~ C ~ m ~ m w $ ~ ~
.. . . .. . _ .

~-- 21 1338706 Breast Cancer Cell Binding Range Determination Antibodies were further evaluated for range of breast cancer cell line recogn~tion by 1~munofluorescence assays on 14 breast cancer cell lines. Table 3 below reports the results of these tests for the 5 claimed antibod~es.

~ ~338706 C`l ~ _ C~ o o ~ o ~ -- ~ ~ ~ ~ ~ ~ rl ~ o rl O ~`: O ~ ~ O N ~ C`~
. C.
o -- o o o o ~ ~ ~ ~ ~ ~r ~ ~ ~ ~ ~ ~ ~ ~ ~ o o c~ ~ ~ ~ ~ ~ o ' X
o o -- ~ ~ ~ o o c~ ~ o ~ o ~ o ~ ~ ~ o ~ c~ o ~ ~ o Cq ~') ~
¢ ~ O O C`~ O O O C`l ~ O ~ O O O Z O
'~ ¢ _ 6 ~0~ 000~00~ 0~ ~~~
o _ ~; ~ ~ ~ o o _ o ~ ~ o o ~ ~t ~ ~ ~ ~ ~ ~ o ~ ~ o ~ ~ ~ o -- ~ ~ ~ ~'I
,1 'I~
E- ~ ~ o ~ ~ o o o 3 ~ ~ o o ~ o o o o o ~ ~ ~ ~ ~ ~ o o o o o ~ o o ~ ~ o o o ~ o m ¢ ~ ~ ~ 7 o o o o o o o c~ ~ ~ ~ ~ o ~ o ~ ~ o ~ ~ ~ ~ ~ o o o c ~ e~ ~ ~ ~ t t ~ ~ ~ ~ ~ ~ ~ ~ c l o I ~ I ~ ~ I
¢
¢ ~
o ~ o o o c~l ~ o o o o o c~l ~ ~ ~ o o ~
o o ~ ~ C`~ ~ -- ~ ~ ~ . ~ ~ ~ ~ o ~ ~ ~ o ~ ~ ~ o o ~ ~

a: ~ O rl ~ o o o ~ ~ ~ ~ ~ ~ r~ ~t ~ ~ ~ ~ O c~l ~ O ~ ~ ~ O O
~ ~ ~ ~ ~ ~ ~ ~ ~ ~ o o C~ ~ o -- C.~ ~ ~ o ~ ,~
. . ~

23 13387~
Non-Breast Cancer Binding of Imaging Monoclonal Antibodies F~nally, the ~ntibodies were tested by ' .~",2;0.~idase stain~ng on eleven non-breast malignancies. The results for the claimed ant~bodies are reported in T~ble 4 below.

13387~

.
O ~ _ o -- o o o o ~ o o _ -- C`l C`l -- -- ~ -- -- o o ~ -- o o C`~ o o o C`l o o o o o o o o o o o o o o o o o o ~ -- o o C`~ o o o o o o o o ~1, 0 0 0 0 0 0 ~ -- -- O O O O O -- -- O O -- ~ -- O ~ O O O O O O O O
.~ ~
o o o o o o ~ o o o -- o o o o o o o o ~ o o _ o o o o o o o o m o~oooo_clo~o oo o oo oc~oo oooooooo ., . ~ O O _ O O ~ O O ~ ~ _ ~ O ~ O O O O O _ O O O O O O O O O O O
~0 ~ ~ O _ O O -- ~ -- _ ~ O -- ~ -- -- -- O -- ~ ~ O ~ C`l O -- C`l O O O O
P~ ~ O O ~ O ~ O O O ~ O _ O O ~ ~-- _ O ~ -- _ O O _ -- O ~ ~ O O _ O ._ ~ U~ ~ O O O ~ ~ ~ ~ O ~ O O -- ~ -- -- O -- -- -- -- O O -- _ -- ~ O O _ O
_ ,1 o o o -- c~l o _ ~ ~ o o _ o c~ o _ -- .-- ~ _ o o o o o -- c~l o o -- c~l C~ ~ _ O O ~ O O O O O O O O O O O ~ O _ _ O O O O O O C`l O O O O ::

~ 25 13387~6 The tumor breast cancer range, breast cancer cell binding range, blood cell binding and select~vity characteristics for the monoclonal ~ntibodies ~ccording to the ~nvent~on are summar~2ed in Table 5.

Imaging MAB Candidates Bl ood Tumor Cel l MAB Cells Range Range Selectivity ''G3 0 1.00 1.00 0.078 C6 0 0.86 0.57 0.063 ~2A1 0 0.33 0.79 0.078 ~3F8 0 0.19 0.71 0.063 ~5E10 0 0.62 0.14 0.070 ~lB4 0 0.67 0.00 0.023 87H7 0 0.95 0.00 0.078 ' 06A10 0 0.86 0.86 0.086 ' 13F1 0 0.14 0.79 0.047 20H7 0 0.67 0.57 0.047 :40A7 0 0.71 0.36 0.070 ' OOF9 0 0.52 0.71 0.031 ' O~E2 0 0.86 0.055 ~1~F3 0 0.86 0.86 0.086 '4-E7 0 1.00 1.00 0.070 ' 5~H9 0 0.92 0.064 "60F9 0 0.52 0.92 0.089 ''66B2 0 0.71 0.83 0.070 ~:7G5 0 O.L3 0.77 0.0 5 ~t qF10 0 0.::: 0. 7 0.0 3 ~,qH9 0 O. '~ O.ql 0.0,6 ' E8 0 0.~:: 0. ;7 0.0~5 C3 0 0.~ 0.~1 0.0'0 ~`i' E Z O 0. ' 0.00 0.023 ''F 0 0.''~ 0.55 0.000 ~ A:2 0 0. ~ 1.00 0.031 L~C:1 0 0. ~ 0.75 0 078 7D- 0 0.~ 0.10 0 039 '~OC9 0 0.'' 0.40 0.008 ~; O 2 0 O.. f O.9t) 0.008 ii~7'3 0 0.: 0.8: 0.070 ~41 8 0 0.:. 0.6~ 0 000 '59~3 0 0. ~ 0.7~ 0 008 '88(~6 0 O.f2 0.8~ 0.016 26 133870~
Antibody Affinity and Antigen Density Several of the claimed ant~bodies were iodinated and tested for binding to MCF-7, CAMA1, SKBR3 or ZR7530 cells. The antibodies were labeled with 125I uslng chloramine T to a specific activity of 5 approximately 10 ~ g. To determine ~ adlochem~cal purity, 100,000 cpm of two of the labeled antibodies in 0.5 ml fetal calf serum was serially absorbed w~th five al~quots of target cells for 15 minutes at 0C (generally 4,000,000 cells per aliquot), and the remaining radioactivity 1n the supernatant after each absorption was lO determi ned.
For measurements of association constants known concentrations of labeled and unlabeled monoclonal antibod~es were incubated with target cells in fetal calf serum for 15 minutes in ice. Aliquots of the cell/antibody mi~ were then counted in a gamrna 15 counter or filtered through Microfold filter plates (V ~ P Scientific) and the filters counted. To account for unbound antibody retained in liquid on the filters, controls containing the same concentrations of antibody but no cells were done in parallel. Association constants and antigen copy number per tdrget are calculated from the affinity 20 test results and are reported in Table 6 below.

TA BLE 6, Affinity and Antigen Copy Nu~ber of InQg~ng MABs MAB s n Ka Cell L~ne ''G3 3700000 9.1x106 MCF7 'A' ~F~`
~"E:O
~'BL
' . H7 113F1 2300000 1.1x109 MCF7 ' 20H7 210000 6.2x106 MCF7 :4DA7 'OOF9 -`03E2 "19F3 "45E7 ' 54H9 60F9 30000 6.0x107 MCF7 ~6B2 80000 2.7x108 MCF7 ~:7G5 3200000 1.6x106 C~MAl '7H9 ~' lE8 ~5' C3 400000 1.4x108 MCF7 ~5-'E 2 --F' 250000 6.8x106 SKBR3 ~''~ 2 470000 1 2X108 MCF7 ~C: 1 390000 4 8x107 ZR7530 -7D.
'~'OC~ 500000 8.2x106 SKBR3 -OE `
~7B

7~1F~
~~ 9E~
7:.8G6 In order to identify the~antigens recognized by the monoclonal antibodies ~ccord~ng to the invention, I ~ipitation of the antgiens was carried out according to the following method.
Eight mm diameter polystyrene balls (Precision Plast~c Ball Co.) were 5 covered with 10~ fuming nitric ~cid in glacial acetic ~cid ~nd were incubated for three hours in a 50C water bath. Following the acid treatment, the balls were rinsed three times with distilled water, covered w~th lX sodium d~thionite in 0.1 M NaOH and incubated three hours in a 50C water bath. The balls were again r~nsed three times 10 with distilled water, covered with 0.1X 1-ethyl-3-(3-dimethylaminopropyl )-carbodiimide (EDAC), 0.2~ suberic acid (suberic ~cid dissolved in dimethylfomamide) and incubated overnight at room t~ ~at~,r~. The balls were rinsed three times with distilled water, and marked for identification.
Purified monoclonal antibodies were diluted 0.2 mg/ml in 2-(N-morpholino)ethane sulfonic acid buffer, and the previously treated and marked polystyrene balls were placed in individual tubes and covered with 450 microliters diluted antibody and 50 microliters of fresh 1~ EDAC. Tubes were capped and i ncubated at 25C for 24 20 hours. Following this incubation, the balls were rinsed twice with PBS and were either used fresh or were stored for several days at ~C
before use.
Freshly labeled target cell extracts were prepared from human breast cancer cell l~nes labeled with 125-I by the 25 lactoperoxidase method of Marchalonis, J., "An Enzymic Method for the Trace lodination of Immunoglobulins and other Proteins", Biochem. J.
113:299-305 (1969), or with 35-S by growth in 35-S methionine. The labeled cells were dissolved in solubilization buffer (1X (v/v) Triton X-100, 150 nM NaCl, 5 nM EDTA. 25 mM Trjs-HCl, pH 7.5). Four parts of 30 labeled extract were mixed in a vessel with one part solubil~zation buffer contain~ng 50 mglml bovine serum albumin, to give a final concentrati on of 10 mg/ml BSA . The bal l s coated wi th monocl ona l antibody were ~dded to the vessel and were incubated four hours on ice with shaking. I~beled ~ntigen was pipetted from the vessel and the 35 balls were rinsed four times w~th solubilization buffer. The balls _ 13387~6 were then removed, placed ~n individu~ tubes w~th 100 microliter Laemmli SDS gel sample buffer, and were incubated three minutes in boiling water. The balls were removed and the samples were run on an SDS gel with appropriate standards.
T pr~cipitation tests on the antibDdies 1ndicated that f~ve of them (454C11, 452F2, 520C9, 741F8, and 759E3) all bind a monomer~c protein of about 200 K daltons found in cancerous breast tissue. Two of the five (520C9 and 741F8), are believed to recognize the same epitope on the 200 K dalton protein. 454C11 and 759E3 bind a 10 second epitope on the same antigen, and 452F2 binds a third epitope on the same antigen. Four of the antibodies (41B4, 87H7, 452E12, 457D7) bound to a 230,000 dalton intracellular antigen. Seven antibodies (2G3, 200F9, 203E2, 245E7, 369F10, 697B3 and 78~3G6) bound to high molecular weight mucins (HMW). T~o antibodies (51C3 and 454A12) bound 1~ to transferrin receptors in the form of a 97,000 dalton antigen.
Neither 451C3 nor 454A12 blocked binding of transferrin to the receptor. The antigen binding characteristics of the monoclonal antibodies according to the invention are summarized in Table 7.

A
~ 30 `1338~06 Ant i gen 2G3 HMW Mucin 32Al 106A10 ~5 K a 113F1 ~0, 60, 100, 200 K
Very Diffuse ' 20H7 HMW Muci n ' 4~7 Glycolipid (pentasaccharide) ''00-9 HMW Mucin 03~2 HMW Mucin '19 3 45~7 HMW Mucin "54 9 '" OF9 55 K b ' 6B2 55 K b ~ 7G5 42 K c ;~'9F10 HMW Mucin ~7H9 40 K
~' E8 ~ C3 ~ransferrin receptor L' 'E12 ''40 K
L F2 '00 K
L ~12 ransferrin receptor ~-LCll ~00 K
4 7D7 ''40 K
5 OC9 ~00 K
6 OE2 42 K c 6~7B3 200 K
7'9E3 200 K
7~8G6 HMW Mucin Different epitope than that bound by 260F9 and 266B2 b ~ Different epitope than that bound by 106,~10; both 260F9 ~ nd 266B2 appear to bind to same epitope c ~ Cross block each other Anti body Isotype Antibody 1sotype was determined as follows: A grid of 5-mm squares is lightly drawn ln pencil on the nitrocellulose sheet and 1-ml droplets of antilsotype ser~ (Litton Bionetics, Kensington, 5 Maryland, rabbit antisera to mouse K, ~ rl, ï2a, r2b, ~3, and chains) are applied so that each row of squares receives one spot of each heavy and light chain reagent. The sheet ~s incubated one hour at room temperature in a moist chamber, r~nsed quickly in PBS-BSA, containing lS (w/v), and left overn~ght ln PBS-BSA at 4C. Strips are 10 cut apart with a scissors and may be stored at 4C in PBS-BSA
containing 0.02~ sodium azide. Alternatively, strips may be air-dried and stored desiccated at 4C. A ser~es of small tubes is prepared containing 3 ml hybridoma culture supernatant or supernatant diluted w~th PBS-BSA. 1:10 dilutions are generally successful; and some 15 supernatants can be diluted as much as 1:200. A nitrocellulose strip is incubated in each tube for one hour at room temperature. The strips are rinsed three times in PBS-BSA and incubated for one hour at room temperature in diluted rabbit ~nti-mouse-horseradish peroxidase. The strips are rinsed twice in PBS-BSA and twice in Tris 20 buffer. The str~ps are placed in Tris buffer containing d~aminobenzidine and hydrogen peroxide until sufficient color develops on the anti-isotype spots (usually 3-4 minutes). The antibody ~sotypes are indicated in Table 8.
, TABLE 8, Isotype of Imaging M~Bs MAB Isotype "G3 Gl ~C6 M
~2A 1 Gl ~3F8 Gl 41B4 Gl 87H7 Gl ' 06A10 Gl 13Fl G3 :20H7 M
'40A7 M
'-OOF ~ ~:
O~E- E:
'~F~ ~:
A'E7 C:
' ~ ~H~ M
~t ~Fg Gl n~t;B2 Gl ~:'G5 Gl F O M
~'`7H~
~''E Gl :C~
~E 2 rl ~'~F' t.~l ~5~A 2 tl ~5~C 1 ~'2A
~57D- Gl 20C 1 Gl li50E'- Gl t'97B~ rl 741F' Cl 759E~ rl ~a.~ c~

Samples of the hybridomas that produce the claimed monoclonal ant~bodies are deposited in the Collection of In Vitro International, 7885 Jackson Road, Suite 4, Ann Arbor, Michigan 48103, USA .
EXAMPLE I
This example shows one method for the labeling of antibodles according to the invention w~th radioisotopes of iodine, either 125-iodine or 131-iodine using a method known to iodinate tyrosine res ~ dues .
Monoclonal antibodies according to the invention may be labeled by the following micro method: 0.1 milligram of the purified monoclonal antibody is labeled with 10 millicurie amounts of 125 iodine as follows: A one inch, 21 gauge needle is inserted partially through the septum of a 3 ml vial and a 3.0 ml disposable syringe 15 barrel packed with glass wool is attached to the needl e. The monoclonal antibody in 0.1 N NaCl preferably not exceeding 0.2 ml in volume, is added with a tuberculin syringe equipped with a 20 gauge needle that has been prerinsed with borate buffer. The sodium 125-iodine solution preferably not exceeding 0.2 to 0.3 ml in volume is 20 added with a syringe attached to an 18 gauge needle pre-rinsed with buffer. The mixture is agitated briefly to mix the protein ~nd 125 iodine solutions. Final dilution of the iodine chloride is made by m~xing 0.2 ml of 125-iodine chloride at approximately 1.25 x 10-5 molar (M) wlth a specific activity of 10 millicuries per mole. After 25 approximately one minute, an excess of 6.25X solution of human serum albumin or animal albumin such as bovine serum albumin is added to the solution. Ihe labeled antibody is passed through an appropriate column to remove unbound radioactive iodine; an ion exchange resin or gel filtration medium such as Sephadex~G-25 may be used. For a 30 Sephadex column purification, after passage of the labeled antibody through the resin at the rate of about one ml per minute,- the resin is rinsed with an additional 1 to 1.5 mls of the abo.L lloned human albumin solution.
/ rQde I`ta r~<

~ 1338706 EXAMPLE II
The monoclonal antibodies according to the invention may also be iodindted by linking agents. This example describes the radioactive labeling of the monoclonal antibody w~th ~n iodinated 5 imidination reagent. The imido ester methylparahr~lG,.~b_~,zl.,11date HCL
(MPHBIM) is synthesized according to the method described by Wood et al., "The Radioactive Labeling of Protein With an lodinated Imidindtion Reagent", Analytical Biochem. 69:339-349 (1975). The MPHBIM is iodinated as follows: 3.7 ml of MPHBIM is dissolved in 1 ml 10 of 50 millimolar mM sodium borate buffer pH 8.5 to obtain a 20 mM
MPHBIM stock solution. 1.0 ml of 40 mM sodium iodine followed by 10 microliters of sodium iodide-125 solution having a specific activity of approximately 2 millicuries per mole is 2dded to one ml of the MPHBIM stock solution. One ml of 40 millimolar chloramine T is added 15 with rapid mixing. The mixture is kept for approximately 15 minutes at 20-22C and then 0.1 ml of 1.0 molar ~-mercaptoethanol is added to reduce the chloramine T and residual iodine. The pH of the solution is subsequently lowered toward neutrality by adding 20 microliters of 1.0 molar acetic acid and a floculant white precipitate forms.
20 Unreacted MPHBIM iodide and chloramine T remain soluble. The precipitate of the iodinated am~no ester is collected by centrifugation at 10,000 rpm for five minutes, dissolved in two mls o~
50 rM sodium borate buffer, pH 8.5 at 37C. Iodination of the antibody is carried out as follows: Twenty milligrams of purified 25 antibody is suspended in one ml of 4 mM lodinated linker, 50 mM sodium borate buffer at pH 9.5. The reaction is carried out at 37C for a period of time sufficient to achieve the desired amount of binding.
Under these conditions the radioactive label is incorporated onto the antibody at a rate of about 1-2X per hour with a maximum incorporation 30 of approximately 30X of the iodine-125 label. Unreacted linker may be removed by dialys~s against 0.15 molar sodium chloride containing a 5 mM sodium phosphate at a pH 7.4.

35 133~706 EXA MPLE I I I
Labeling of the Monoclonal Antibody with Chelating vcroups-DTPA
The ,,,vl,oclol-al antibodies may be labeled with 111-ln using the chelating agent diethyl~ llid~ vlJellLdavv ;v acid (DTPA) anhydride according to the method of Hnatowich et al, Science 220 613-615 (1983) Antibody 11 3F1, is prepared at 11 milligrams per ml dialysed into NaHCO3 at pH 7 One mg DTPA cyclic anhydride was dissolved in 10 ml CHC13 Forty ~r1 of this solution was delivered into 5 mlglass test 10 tube and the CHCl3 was evaporated wlth a stream of N2. One-hundred microliters of 113F1 (1.1 milligrams protein) was added to the tube containing 4 micrograms of anhydride and the tube was vortexed briefly. After one minute, 5 microl~ters of 111-1n (having a specific activity of about 3.28 x lOlU cpm/ml) in 0.5 molar sodium acetate at 15 pH 5.8 was added. Two PD10 (Pharmacia) columns were prepared with 20 milliliters of phosphate buffered sallne lX bovine serum albumin. The samples were run on the PD10 columns with 2.2 milliliters of void volume. A 2.5 milliliter protein peak and 2.5 milliliters small molecule peak were found eluting with the PBS lX BSA. The control was 20 a 100 microliter sample of 113F1 together with 4 microliters of one microgram/microliter DTPA, not the anhydride, and 5 m~croliters of 111-In. The DTPA anhydride labeled 113F1 protein peak contained 75Z
of the counts and the small molecule peak/fraction contained approximately 25X of the counts. In the control approximately 92X of 25 the counts remained in the small molecule fraction.
EXA MPLE I V
Labeling of Antibody With Var~ous Activities of Indium Monoclonal ~ntibody 113F1 from the previous experiment was diluted in 50 millimolar NaHC03 pH 7 to concentrations of 100, 10 and 30 1 micrograms per 100 ILl. One hundred l~l of each dilution of 113F1 was added to 4 ~9 DTPA anhydride in a glass tube as in Example III. 111-ln was dlluted in 0.5 molar sodium acetate pH 5.8 to 100 microcuries per 10 Ill. One hundred m~crocuries 111-In in 10 microliters solution was added to the tubes contatning the antibody. The mixture was 36 ` 13~8706 treated as in Example llr. Seventy-six pertent of the counts were found in the prote~n fraction of the one m~crogram per 100 microliter dilution and 86S of the counts were found in the 100 microgram per 100 microliter d~lution.
EXAMPLE V
Labeling of Antibody 245E7 with lll-lndium This example shows the labeling of another antibody according to the invention w~th lll-ln.
1 mg DTPA cyclic anhydride was dissolved in 10 mls dry CHC13. 40 11l of the dissolved DTPA anhydride was placed in a S ml glass tube and was evaporated with N2 to y~eld about 4 119 DTPA cyclic anhydride coated on the inside of the tube.
100 11 of antibody 245E7 at a concentration of 15 mg/ml in 50 mM ~aHC03 pH 7 was added to the DTPA cyclic anhydride. The tube 15 was briefly vortexed and left to stand for about one minute forming the DTPA-245E7 complex.
Five test samples were made up as follows:
(1) 10 ~1 DTPA-245E7 complex in 90 ~1 NaHC03 (2) 10 ~L1 DTPA-245E7 complex in 90 1~l NaHC03 (3) 10 1~1 245-E7 in 90 1ll NaHC03 (4) 10 1-l 245E7 in 90 1~1 NaHC03 (5) 10 1~1 245E7 in 90 ~1 NaHC03 and 0.4 1~9 DTPA
not anhydride form.
lll-ln was diluted to a specific activity of 100 cpm/lll in 25 0.5 sodium acetate pH 5.8. 10 ml of this 111-1n solution was added to each tube. 10 ~9 DTPA in the non-anhydride form was added to tubes 2 and 4. The contents of each tube were run on a PD10 col umn equilibrated with NaHC03 as in Example 111. Samples ~ere collected and the protein and small molecule peaks were counted in a liquid 30 scintillat~on counter using conventional methods. The results are shown in Table 9.

. ~ 13387Q6 Tube Fraction CPM X Total Counts ~P 3263 77 +SM 964 23 Protein + Smal l mol ecul es The results indicdte that 77X of the 111-In bound to DTPA labeled 245E7. Subsequent addition of excess free DTPA does not remove indium from the DTPA-245E7 antibody complex. Sample 3 shows that indium does not non-specifically bind to the antibody in any appreciable amount;
5 however, the indium appears to be reta~ned on the column rather than eluting with the small molecule fraction. DTPA added before or after i ndi um resul ts i n the i ndi um el uti ng i n the smal l mol ecul e peak .
E~MPLE VI
Uptake of 111-Indium Labeled Monoclonal Antibodies to Breast Tumor issues This example shows that 111-In labeled monoclonal antibodies are efficiently bound by human breast tumor tissues.
Six anti-breast cancer tumor monoclonal antibodies 113F1, 245E7, 260F9, 280D11, 2G3, 266B2, 3nd a negative control MOPC21, were covalently linked to DTPA anhydride by the method described in Example -15 III above. The ant~body-DTPA complex was radiolabeled by chelation w~th 111-In at a speclfic activ~ty of about 1 1l C~tllg. The 111-In labeled antibody was purified when necessary on a 0.4 x 17 cm column of Sephadex G50 to a radiochemical pur~ty of about 90X. Two non-breast spec~fic anttbodies, antt-carcir~l ~ y~ric antigen monoclonal ~nt~body (anti-CEA) obtatned from Medi-Physics, Emeryville, Caltfornia and anti-prostatic actd phosphatase anttbody (anti-PAP) obtatned from New England Nuclear Corpordtton, Boston, Massachusetts) were labeled 5 tn the sdme manner as the antt-breast cancer tumor ~nttbodies and served as posttive binding controls. Human breast and colorectal tumor tissues were obtained tmmedtately after surgery and were placed ln fresh Eagles Minimal Essential Media (MEM) supplemented with 10X
fetal calf serum, non-essenttal am~no actds, glutamine, penicillin and lO streptomycin (MEM) for transportation. Fresh tissue was used within three hours of recetpt while c, jop, ~;L ~d tissue was matntained in MEM at -70C. The ttssues were sectioned manual ly with surgical blades into 1.0 ~ 00.2 mm cubes and checked for size accuracy using an ocular micrometer. Using stertle techniques, the tissue cubes were 15 transferred to a 96-well microttter plate containing 200 ~l of MEM and either 1.0 or 10 ~l9 of 111-In-labeled antibody. The tissues were tncubated from 1-24 hours at 37C in a 5~ C02 water-jacketed incubator. Follow~ng tncubation, the media was carefully removed using an automatic ptpetter wtth minimal disruption of the tissue, and 20 fresh media was added. Following tncubation for an additional 20 minutes, the med~a was again replaced and the tfssue was incubated for another 20 m~nutes. After this last wash, the media was removed and the tissue transferred to ~ dry tared weighing paper. Tissues were dried at 70C for 20 minutes and were then weighed and placed in test 25 tubes for counting in a NaI well counter. The results are reported as the percent of applied radioactivity in tissue per unit weight of dried tissue. Small differences tn the size of each tissue cube were corrected .
Speci fi ctty of Bi ndi ng To establish that the accumulation of radiolabeled antibody tn tumor ttssue is due to spectftc btnding rather than non-specific adsorption, radiolabeled antt-CEA and anti-PAP antlbodies, used as controls, were tncubated with fresh and cryopreserved human colorectal tumor tissue whtch expresses CEA. Figure 1 shows the percent 39 1338~06 incorporation of radioactivity YS. incubation time at 37C for both ~ntibodies, each at 1 ~9 and 10 I~g/well, for one tumor tissue.
At the 1 ~g/well concentration, the anti-PAP antibody shows little incorporation at any time. The ~nti-CEA ~nt~body by contrast S shows about a 20 fold increased ~ccumulation. At 10 l~g/well, the difference in radioactivity accumulation for the specific and non-specific ~ntibodies is much less indicating saturation of the antigenic sites.
Specific binding of the anti-CEA control antibody was 10 further demonstrated by a competitive binding study. Tumor tissue was pre~ncubated with saturating levels (25 ~9) of unlabeled ~nti-CEA
antibody for 17 hours prior to the normal assay using 1 ~9 of labeled anti-CEA antibody. Control wells did not receive the unlabeled antibody. As shown in Table 10, in the case of the non-specific 15 antibody, there was essentially no change in tissue accumulation of radioactivity with preincubation whereas in the case of the specific antibody, a large decrease in accumulation occurred in the tissue preincubated with the unlabeled anti-CEA antibody.

20 Binding of radiolabeled anti-CEA and anti-PAP to colorectal tumor tissue without preincubation with unlabeled anti-CEA antibody Weight of unlabeled anti-CEA Radioactivity Bound*
Ant~body antibody per well ~1~9) (~) Anti-CEA 0 28.3 1 . 2 Anti-PAP 0 1.3 25 0.8 ~ Mean value (n-3) Selectivity of Binding A p~nel consisting of labeled anti-CEA ~nd ~nti-PAP and the si~ anti-breast cancer tumor monoclonal antibodies ~las tested using two human breast tumor tissues. Replic~te measurements of binding of the same antibody ln the same t1ssue show only small variations, while the varidtion in binding of the same antibody in different tissues or different antibodies in the same t1ssue is far larger, as shown in Figure 1.
The antibody 113Fl showed only modest binding in one of the tumor tissues but showed the highest degree of binding in the other.
Although as expected, the anti-CEA antibody showed the same degree of binding as the anti-PAP antibody in one of the breast tumor tissue, the former showed increased binding with respect to the latter in the 10 other tumor tissue tested.
Also in Figure 1 (left panel) is presented the results of a repeat measurement of the same antibodies and the same tissue analyzed initially (solid bars) and three days later (cross hatched bars).
Although there are slight differentes in the level of uptake of 15 1ndividual antibodies, the order, ranked according to antibody accumulat10n, is unchanged.
EXAMPLE Vl I
Nineteen 8 week old female nude mice were implanted with MX-1 tumors subcutaneously in the r1ght dorsal flank. The mice were 20 furnished with food and water ad libitum. At 14 days after implant when the subcutaneous tumors had reached a size of approximately 0.5 cm3, the water was replaced with water conta1ning 0.1~ Kl.
Monoclonal antibody 260F9 was labeled with 125-I
1,3,4,6Tetrachloro-3a,6a-diphenyl glycouril (lodogen~) as follows. lO
25 ~l of Iodogen~ was placed in a sterile glass test tube and 1 UCi of 125-1 as Nal salt with a specific activity of about 17 Ci/mg tNew England Nuclear) was added to the iodogen. Monoclonal antibody 260F9 in phosphate buffered saline, wlthout az1de was added to the lodogen' 125-1 to label the antibody at a specific act1vity of 5 IICi/l~g 30 ~ nt i body .
Monoclonal antibody MOPC21 was labeled in the same manner and served as a control. Approximately 2 ~9 of labeled antibody conta1ning approx1mately lû ~Ci 1-125 was administered to each 41 ~38~06 mouse. The labeled ~ntibody was administered in a volume of approx~mately 0.1 ml PES containing 17l 55A, via the mouse tail vein.
Four days ~fter administr~tion of the labeled antibody the mice were exsanguinated by eye puncture. The blood was heparln~zed, centrifuged 5 and the blood plasma was retained. The organs were disected, chopped ~nto approximately 1 MM3 pieces and were washed in saline to remove excess counts. The chopped tissue was weighed and the radio ~ctivity was measured in an LKB gamma counter.
The tissues of six mice treated with iodin3ted MOPC21 served 10 ~s controls. The tissues of 13 m~ce treated with 1-125 labeled 260F9 served as test tissues. Counts per minute per gram (cpm/gm) tissue or tumor were determined and an index of uptake was determined by the ratio of cpm/gm tumor to cpm/gram organ (T/O ratio).
Table 11 shows the T/O ratio for 260F9 at 10 ~Ci 1-125 for 15 MX-l tumors in each animal tested. Table 12 shows the T/O ratio for MOPC21 at 10 IlCi 1-125 for MX-l tumors in each anim31 tested. Table 13 shows the mean and standard deviation for all tested animals. Figure 2 i8 a graphical depiction of Table 13.

~ 42 13387~
~ o o o æ o g ~D o N N æ N
D O ~D O
~ _ N N
N I ~ g o O O O O O O
_ o r~
O
æ ~ . ~ ~ O 0. 0 ~ ~ O O~ ~ ~ O ~
C~ N ~ o~ o ~ ~o~ o oO
~n .....
._ ~ N _ _ _ _ N
O0 ~ ~ ~ N U') 1` ~ O
D 0. C~
~, NO _ U'~ N
0~1~ _ U~ ~ ~ O ~ O
O~ ~ _ ~ O ~ ~ ~ ~ ~
~O ~ -- ~ ~t N
g I G ~ ~ ~ o o cr~
O ~e I N _ _ ~ _ N 1` 3 ) ,,t, O
1~ 1 N O N _ ~ O
_ ~ N N
N ¦ N In ) -- ~ _ _ ~ N
~ o ,o , ~ Uo~ O
N ~ N ~ ~ d ~: ~t N '- _ ~ ~ O CC
N ¦O g -- ~e> ~. -- U- O O
C 1`~ 1~) N 11- O _ O ~ ~
_ ~ _ _ _ _ U`) N
1~ ~9, ", C
~ ' ~ ~ ~L ~ C -- I~J

43 13~7~6 T/O Rat~os of MOPC21 at 10 ~Ci - MX-l Tumors ~270#312 #394 ~375 ~1376 ~535 Plasma .36 .30 .17 .14 .15 .26 Ribs4.85 5.72 1.86 2.20 2.00 2.89 Lungs 2.70 3.37 2.90 1.70 1.00 1.18 Liver 3.71 2.75 1.88 1.50 1.40 1.86 Spleen 5.46 7.66 2.43 1.70 2.00 2.89 Kidney 3.96 2.30 2.16 2.00 1.20 1.86 Heart 3.15 2.42 1.30 1.80 1.00 2.00 G113.25 8.99 3.70 3.90 4.70 8.67 Carcass 3.60 4.30 3.00 2.60 2.00 2.89 Tumor/Organ Rat~os Mean Std. Dev. Mean Std. Dev.
Pl asma 2. 70 1. 89 .23 .09 Ribs 28.92 14.29 3.25 1.64 Lung 10.18 5.60 2.14 .98 Liver 12.22 10.15 2.18 .89 Spleen 8.79 3.60 3.69 2.36 Kidney 13.47 9.39 2.25 .92 Heart 19.30 13.23 1.95 .78 Gl 41.26 25.34 7.20 3.78 Carcass 23.40 19.56 3.07 .80 EXAMPLE V I I I
Prior to exsanguination two mice treated with labeled 260F9 and one mouse treated w~th labeled MOPC2~ according to Example VII
were imaged using a Searle Fho-gamma' camera ~ith a pinhole 5 collimnator. Raw ddta were collected and computor enhanced. The images of the treated and control mice are shown in Figure 3.
In the first line of Figure 3, from left to right, are images of a tumor bearing mouse treated with 125-I-labeled 260F9 prior to surgical removal of the tumor, raw data; a computor-enhanced image 10 of the same mouse; an image of of the mouse, post surgical, raw data;
and a computer-enhanced image of the same mouse, post surgical. A
prominent area of local~zation of detectable radiation is found in a pos~tion corresponding to the MX-1 tumor, on the dorsal right flank of the mouse treated with 125-I-labeled 26ûF9.
In the second line of Figure 3 from left to right are images of a tumor bearing mouse treated with 125-I labeled MOPC21, an antibody not specific for the tumor used as a control, raw data, and a computor enhanced image of the same mouse. There is no correspondi ng area of localization of detectable radlation on the dorsal right flank 20 as in the mouse treated with 125-I labeled 260F9. Furthermore, the distribution of label fn the control mouse appears to correspond to the distribution of label in the post-surgical mouse treated with 125-I-labeled 260F9.
In the third line of Figure 3, from left to right are images 25 of a tumor bearing mouse treated with 125-I labeled 260F9, pre-surgical, raw data, ~nd a computer enhanced image of the same mouse.
A prominent area of localization of detectable radiation is found on the right dorsal flank of the mouse in a position corresponding to the MX-1 tumor.
In the fourth line of Figure 3, from left to right are images of the tumor bearing mouse from line 3, post surgical, raw data; a computor enhanced ~mage of the same mouse, post surgical; an image of the tumor excised from the same mouse, raw data and a computer enhanced ~mage of the same excised tumor. A significant ~'rra~ l~rk ... . .. . .. .

45 13~8~
amount of the labeled tumor-specific antibody i5 shown to have localized ln the tumor. The amount of detectable tumor-specific antlbody remalnlng in the mouse post surgically appears to be less than the amount of detectable antlbody ln the control mouse ln line 5 two in thls llmited sample.
The monoclonal antlbodies according to the lnvention, after derivatization with a labeling moiety, have a number of uses. Ihe lmmunoimaging monoclonal antibodies may be used ln diagnosis of primary malignant breast tumors. Patients presenting with masses 10 lndicating a posslbllity of malignant breast tumors presently routinely undergo a ser~es of diagnostic mammographic examinations.
In addition to mammography, the lmmunoimaging antlbodies according to the lnvention may be administered subcutaneously or intravenously to determine whether the mass is positive for uptake of the labeled 15 antibody according to the invention. Accumulation of the labeled antibody would serve as 2n additional indication suggesting the need for a biopsy or more extensive surgical intervention.
The labeled monoclonal antibodies according to the invention also have a clear use in assaying the clinical prognosis of patients 20 who have had mastectomies or lumpectomies for removal of malignant breast tumors. Conventionally, the axillary lymph nodes of such patients are disected to determine the extent of dissemination of the mal~gnancy. Under current practice, patients with positive nodes receive a course of adjuvant chemotherapy. Axillary node sampling is 25 an invasive procedure requiring general anesthesia. It entails all the risks of any major surgical procedure including infection and reaction to anesthetics, and requires a significant post operative period of pain, recovery and healing.
The monoclonal antibodies according to the lnvention and the 30 derivatives thereof, can be used as a non-invds1ve method for determining the nodal involvement of a breast malignancy and ~ay serve as an adjunctive procedure to conventional nodal dissectlon or as a replacement therefor.

46 133870~
The utility of radiolabeled monoclonal antibodies has been shown, ~t least in a preliminary manner in a number of clinical studies. McKenzie et ~l. "Immunoscintigraphy for Detection of Lymph Node Metastases From Breast CancerU Lancet No. 8414:1245 t1984) have 5 shown that subcutaneous interdigital lnjection of an I-131 labeled monoclonal antibody specific for a human malignant breast tumor, can be used to confirm the presence of metastases in patients who were already suspected to have tumors involving axillary lymph nodes, and to detect tumors in lymph nodes where the presence of tumor had not 10 been suspected. Using a Toshiba GCA402 gamma counter camera and a high energy parallel hole collimator-computerized equalization with an Informatek Simes 4 computer, at 24 hours post-injection, immunoscintigraphy was more sensitive than conventional clinical examination for the detection of metastases in draining nodes.
Breast tumor localization with the labeled derivatives of monoclonal antibodies according to the invention by intravenous administration, is also a clear alternative to the subcutaneous administration route. In this method, the radiolabeled monoclonal antibody is introduced into the patient in a solution appropriate for 20 IV administration such as 0.15 M NaCl with lX human serum albumin.
The radiolabeled monoclonal antibody is injected preferably using a venous catheter in a volume of saline over an approximately 30 minute per i od .
In both the subcutaneous and intravenous administration 25 methods, the patient is tested for allergy to the normal antibody of the animal from which the monoclonal producing hybridoma was produced. In general, if the monoclonal antibody is derivatized with a radioisotope of iodine, the patient is pre-treated with Lugols iodine-solution to block thyroid uptake of 131-I, and premediated with 30 promethazine and prednisolone before administration of the lmmunoimaging monoclonal antibody. Patients are scanned over a period of hours to days after administration of the immunimaglRg monoclonal antibody. Scanning methods for radioisotopic lmaging including appropriate control proc~lu e; such as subtraction analysis with non-35 specific antibody are known to those skilled in the art of nuclear ~;--47 133~7a6 medicine and include computer assisted photos~r- ing and computer assisted tomoscintigraphy.
Other clinical uses of the labeled monoclonal antibodies according to the ~nvention are clear to those skilled 1n the art of 5 breast cancer pat~ent management. Such uses ~nclude the use of the labeled monoclonal ~ntibodies to monitor the response of metastatic tumors to therapy using various therapeutics including ch ~ a~ ics, immunoto~(ins, I - 'I LJ or lymphokines.
The labeled monoclonal antibodies according to the invention 10 may also be used to detect the presence of life endangering highly morbid metastases at a time prior to their symptomatic manifestation early enough to permit preYentive or ameliorating radiotherapy. The most highly selective antibodies according to the invention may also be labeled to determine the distribution or localization or lack 15 thereof of the monoclonal antibody in the normal tissues of patients thus providing a basis for identifying breast cancer specific monoclonal antibodies that may be advantageously used as components for antibody based therapeutics such ~s immunotoxins or immunodrugs for the treatment of malignant breast tumors.
These and other aspects of the invention will be apparent to those ordinarily skilled in the art to which this application pertai ns .
The monoclonal antibody-producing hybridomas listed below were deposited with the American Type Culture Collection (ATCCj or 25 Invitro International Inc. (IVI) under the terms of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure and the Regulations t~ ~ ' (Budapest Treaty). This assures maintenance of the viable culture for 30 years from date of deposit. The hybridomas 30 will be made available by ATCC or IVI under the terms of the Budapest Treaty, and subject to an ~greement between applic~nts ~nd ATCC or IVI
which assures unrestricted availablity upon issuance of the pertinent U.S. patent. Availability of the deposited strains ~s not to be construed as a license to practice the invention in contravention of 48 13387~
the rights granted under the authority of any go..., ~ in accordance with its p~tent laws.
Each hybridoma designation listed in the left column co~ (~s, . ' to the monoclon21 ant1body produc~ng the des~gn2ted 5 monoclonal antibody.
~ .
Cetus ~ IVI A~cession No. ATCC Accession No.

lQ 41B4 IVI-10057 IIB 10786 106A10 IVI-10~060 HB 10789 4211~8 IVI-10064 HB 10793 351~10 IVI-10067 HB 10796 219P3 . IVI-10072 HB 10801 387H9 IVI-10073 lE~B 10802 452B12 IVI-10074 ~B 10803 2s 457D7 . IVI-10076 ~B 10805 -~ 452F2 IVl-10082 HB 10811 Cell Line Deslgnation ATCC Access~on Number 35 G3 HB--4'~1 ~F8 H~-''6'17 _3F1 H'-'4'~0 ~SE7 H~ L:l ' fi6B2 H ~ h~;
_:7G5 H''-' 3t 9F10 HB-~F.~
4 4C11 HB-P.~L
E

Claims (13)

1 A murine monoclonal antibody suitable for imaging breast tumors and selected from those obtainable from ATCC-HB10785, ATCC--HB10795, ATCC--HB10796, ATCC--HB10786, ATCC--HB10788, ATCC-HB10789, ATCC--HB10790, ATCC--HB10798, ATCC-HB10791, ATCC-HB10799, ATCC--HB10801, ATCC-HB10792, ATCC--HB10802, ATCC-HB10793, ATCC--HB10810, ATCC-HB10803, ATCC--HB10804, ATCC-HB10805, ATCC-HB10806. ATCC-HB10808, and monoclonal antibodies that compete for binding to an antigen bound by any of said specific antibodies.

2. An immunoimaging agent comprising:
(a) a monoclonal antibody of Claim 1, and (b) a detectable label.

3. An immunoimaging agent of Claim 2 comprising:
(a) a monoclonal antibody as defined therein, and (b) a detectable label conjugated thereto

4. An immunoimaging agent of Claim 3 wherein said detectable label is covalently bound to the monoclonal antibody, or is bound to a linker that is bound to a monoclonal antibody, or is conjugated to said monoclonal antibody by a chelating agent.

5. An immunoimaging agent of Claim 4 wherein said detectable label is bound to the monoclonal antibody using N-chloro-p-toluenesulfonamide or tetrachloro-3.alpha.,

6.alpha.-diphenylglycouril, or wherein said linker is methyl-p-hydroxybenzimidate or N-succinimidyl-3-(4-hydroxyphenyl)propionate, or wherein said chelating agent is diethylenetriamine-pentaacetic acid anhydride or ethylenetriamine-tetraacetic acid.

6. An immunoimaging agent of any one of Claims 2, 3, 4 or 5 wherein said detectable label is selected from:
(a)fluorchromes;
(b)radioactive isotopes;
(c)radioopaque substances; and (d)NMR detectable substances.

7. An immunoimaging agent of Claim 6 wherein said radioactive isotope is 123-Iodine; 131-Iodine; 111-Indium or 99-Technetium.

8. A formulation for imaging breast tumors comprising an immunoimaging agent of any one of Claims 2, 3, 4, 5 or 7 and a carrier suitable for parenteral administration.

9. A formulation for imaging breast tumors comprising an immunoimaging agent of Claim 6 and a carrier suitable for parenteral administration.

10. The use of a monoclonal antibody of Claim 1 in producing a formulation for use in imaging breast tumors in a patient in need of such imaging.

11. A hybridoma producing a monoclonal antibody of claim 1.

12. A method of making a hybridoma of Claim 11 comprising fusing antibody-producing cells from an animal immunized against breast cancer cells antigens with myeloma cells and selecting from the fusion products clones secreting antibody which competes for binding to an antigen bound by a specific antibody as defined therein.

13. A method of making an antibody of Claim 1 comprising culturing a hybridoma capable of producing such antibody.