CELL LINE AND MONOCLONAL ANTIBODY

Abstract: A cell line which produces a monoclonal antibody having a predominant specificity to breast cancer antigens.

Description: TITLE: CELL LINE AND MONOCLONAL ANTIBODY Background of the Invention This invention relates to a new cell l ine and . t monoclonal antibodies produced thereby. The invention als relates to a method of detecting breast cancer using th antibodies embodying the invention. Antibodies are primary components of immune defenc systems and are made by the white blood cells (lymphocytes in response to the presence of foreign material such a bacte i , vi us, tumor cells or even inorganic che icals Such foreign material, known as antigens, are chemicall highly identifiable by means of the specific protein produced thereby. In general, antibodies, which are produced by th lymphocytes in response to the presence of antigens, are Y shaped protein molecules, each of which can bind uniquely t the antigen and render same inert. Mammalian antibod defence systems are highly flexible and it has bee estimated that the average person can make, antibodies t more than a million different foreign molecules. Familia antibodies take the form of antiserums such as snake o spider venom antiserum. Other examples include anti-tetanu and anti-rabies antiserums, which are used in the treatmen of such diseases. These antiserums are traditionally made by injectin forms of t.he various disease-carrying micro-organisms (th antigens) into domestic ani als, such as rabbits, sheep, horses and goats. The selective and specific bindin capacity of antibodies has made them- useful for anothe purpose - the detection of very small quantities of foreign molecules or antigens in blood, body fluids and tissues. Antibodies have been used to diagnose disease since thei discovery in the late 19th century, at first in relativel crude systems, but with increased sophistication until man millions of diagnostic tests are carried out annually at the time of writing. Until recently however the full potential of antibody based diagnostic systems has not been real ized. This has been due mainly to the method ' of production as each antiseru produced by an animal is a complex mixture of many antibodies, only a tiny fraction of which is relevant to the required purpose. In addition, no two batches of antiserum are every the same as no animal responds to the introduction of antigen in the same way twice. However in 1975, Kohler & Milstein made a breakthrough in the- United Kingdom in antibody production, which has had an enormous impact throughout biology and medic ne. These scientists discovered a way to make unlimited quantities of identical an ibody molecules from a single, line or clone of cells in a test tube. These dentical molecules were called monoclonal antibodies and it is the use of one of these monoclonal antibodies that is the key to the present invention. Such antibodies exploit the remarkable specificity of the immune system, and bring to fields of medicine and biotechnology a degree of specificity that had previously been impossible to achieve. Monoclonal antibodies are typically produced by using a cultured cancer cell (a myeloma) and a living cell taken from the spleen of a mammal (for example BALB/c strain of mouse). Preferably the spleen cell has already been primed w th an antigen and is already producing a specific antibody. The new cell thus formed is called a hybridoma. The hybridoma displays the characteristics of both parent cells, in that it produces the same antibodies produced by the parent spleen cell and also exhibits the same vigorous growth, production and longevity characteristic of a cultured cancer cell. Hybridomas formed by such fusions are cultured and cloned, and clones which produce antibodies demonstrating a specificity for a desired antigen are selected and cultivated. In this way, an ample supply of a monoclonal antibody specific to the antigen used to immunise or "prime" the parent spleen cell can be obtained. The hybridoma is thus a highly sensitive diagnostic agent for the antigen in question. It is anticipated that the excellent specificity and ready availability of large quantities of monoclonal anti bodies will cause a revolution in immunodiagnostic testing procedures ithin a relatively short time. The abi lity to obtain pure (monoclonal) antibodies specific to individual antigens has resulted in a p oliferation of the use of monoclonal anti bodies. Appl ications i nclude the testing and/or treatment agai nst many antigens derived from bacterial, viruses, hormones, parasites and even various chemicals, although emphasis to date has been placed on the diagnosi s of a va iety of bacteria and viral diseases, on blood and tissue typing, and on assays for several hormones and enzymes whose presence in blood or urine can itself be used as a diagnostic. The appl ication of monoclonal antibody techniques to bi otec hnol og ic al research has been wel l described in the l iterature, including Kohler et al in NATURE, Vol 256,495/497 (1975); Walsh in NATURE Vol 266,495 (1977); R.L. Gatz et al in BIO/TECHNOLOGY 337 of June 1983; L.A. Herzenberg et al in I M U NOGE N ET I CS 1982; George S. Eisenbarth in ANALYTICAL BIOCHEMISTRY 111, 1/16 (1981); U.S. Patents Nos. 4,172,124 and 4,359,457; and Austral ian Petty Patents Nos. 529,210; 530,850 and 530,851. Prior to this invention however, it was not known whether specific monoclonal antibodies could be used to test human blood serum or other secretions for breast cancer. Previously it had been the practice to test suspected mammary tissue that had been removed surgically from the patient. A number of monoclonal anti bodies have now been made which react primarily with human carcinoma of the breast, but also react with other tumours and have a variable reaction with normal breast tissues (see for example Foster et al, Virchows Arch (Pathol. Anat.) 1982; Vol 394, pages 279-293; Papsidero et al. Cancer Research 1983; Vol 43 pages 1741 -1747; Schlom et al, Proc. Natl. Acad. Sci. 1980; Vol 77, pages 6641-6645; Ceriani et al. Somatic Cel l Genetics 1983; Vol 9, pages 415-427; and Thompson et al, J. Natl . Ca ncer In st. 1983; Vo l 70, pages 409-419). Unfortunatel , none appear to be entirely speci fic for carcinoma of the breast and indeed no monoclonal antibody has been described which has been conclusively proven to be tumour specific for human carcinomas. For diagnostic and possibly therapeutic purposes, it is therefore desirable to have a panel of antibodies which in toto define tissue and/or tumour specificity. Brief Summary of Invention This invention provides a new cell line, which has been designated 3E1-2, which produces a monoclonal antibody having a predominant specificity to breast cancer antigens. The invention also provides a monoclonal antibody having a predominant specificity to breast cancer antigens. The invention also provides a method of detecting breast cancer in blood serum secretions or tissue comprising the step of applying to an immunoassay containing the blood serum, secretion or tissue to be tested a monoclonal antibody having a predominant specificity to breast cancer antigens and detecting the presence or absence of a reaction in said immunoassay. The invention also provides for a continuous cell line identified as clone 3E1-2 which produces breast carcinoma antibodies in vitro in hypoxanth ine-aminopterin-thym idi ne med um compri si ng a f used cel l hybrid of human adenoc ar ci noma-pri med BALB/c mouse spleen cells, and NS-1 mouse myeloma cells, said antibodies reacting with a breast tissue antigen. Brief Description of the Drawings Figs. 1 and 2 are graphs showing the results of serum inhibition study conducted on a number of patients. General Description of Preferred Embodiments Monoclonal antibody 3E1-2 is produced against human carcinoma of the breast by the wel l-known method of immunizing BALB/c mice with breast cancer cel ls and performing a fusion of spleen cells from the immunised mouse with NS-1 myeloma cell line using polyethylene glycol as the fusing agent. Antibody 3E1-2 is of the IgM class and is detected using an i mmunoperoxidase method to stain cancer of the breast cel ls. Breast antigens are present in significantl greater than normal amounts in the blood serum, secretions or ' tissue of patients with carcinoma of the breast. The antibody reacts very strongly with carcinoma of t-he breast antigens, and weakly or not at all with normal breast tissue. The antibody the subject of this invention is uni formally negative with the surface of sime in vitro cancer cell lines. However, a 100% positive result has been found on breast cancer tissues. Some positive results were also derived from other cancer tissues such as lung or colon; however, the strength of the reaction was substantially less than the results derived from the breast cancer tissue. Positive reactions are obtained on formation fixed breast cancer tissue using the i mmunoperoxidase method so the antibody provides a useful reagent for histological diagnosis of breast cancer. Antibody 3E1-2 therefore provides a highly satisfactory testing medium on tissue and blood serum for breast cancer using any suitable technique such as ELISA or radioi mmunoassay, both of which are well- known and require no further description. Since the antibody is effective in serum testing, one effect of the method of this invention is that it will be no longer necessary to physically remove tissue from the breast to test for cancer. The test may be carried out on blood serum or other secretions and provides substantial savings in time and cost, as well as providing a simple diagnostic procedure suitable for general practitioners. Further, tests previously produced to test mammary tissue for breast cancer were notoriously unreliable. Lack of sensitivity and specificity often lead to false results, either positive, or regretably negative. The method of the present invention provides results which are substantially more accurate. This is due to the high specificity of the antibodies, produced by the 3E1-2 cell line and extremely high sensitivity of those antibodies to breast carcinoma antigen. As will be apparent from the above, one major feature of the invention lies in. that a breast cancer test can be carried out using blood serum or other secretions as well as on tissue surgically removed from a suspect breast so that * * iOTk nη_ι p m«u v -r y * m OMPI attractive alternatives to tissue testing are provided. Detailed Description of Preferred_ Embodiments Abbreviations Used ABA Azobenzenearsonate BSA Bovine Serum Albumin CEA Carcineombryonic Antigen CPM Counts per minute DMEM Dulbecco's Modified Eagles Medium EDTA Ethylenediamine tetra-acetic acid FCS Foetal Calf Serum HAT Hypoxanthine, Aminopterin, Thymidine medium HCG Human Chorionic Gonaditrophin HLA Human Leukocyte Antigens L15 Leibovitz's medium MFGM Milk Fat Globule Membrane NCS Newborn Calf Serum NS-1 P3-NSl-Ag4-1 PBS Phosphate Buffered Saline PLP Periodate-Lysine-Paraformaldehyde PVC Poly Vinyl Chloride RAMG Rabbit Anti Mouse Immunoglobul in RPMI Roswell Park Memorial Institute medium SAMG Sheeop Anti Mouse Immunoglobul in SRBC Sheep Red Blood Cells TTS Triton X-100, Tris, Saline Materials and Methods Cell Lines All human cell lines were maintained in RPMI-1640 medium containing 10% NCS, penicil lin, streptomycin and glutamine. The mouse myeloma P3-NSI-Ag4-1 (NS-1) was maintai ned in DMEM containing 10% NCS, penicil l in, streptomycin and glutamine. (All media and additiives were obtained from Flow Laboratories, Sydney, Austral ia). Adherent cell lines were harvested after the culture medium was decanted, and the cell monolayer exposed to 0.20% trypsin (com onwealth Serum Laboratories, Melbourne, Austral a) at 37°C, for two minutes, fol lowed by the addition of NCS to neutralize the trypsin. When the cells O PI detached, they were harvested and washed three times i n Leibovitz's medium (L-15) and 0.5% Bovine Serum Albumin (BSA; M i les Laboratories, Ind.) prior to use. To demonstrate that the antigen recognised by 3E1-2 was not trypsin sensitive, tisdsue sections were trated with PBS- versene solution, containing various concentrations of trypsin for up to 30 minutes at 37°C. Fresh Tumour Tissue A breast carcinoma of the ductal type was obtained from a surgical specimen, a single cel l suspension produced in serum free DMEM, centrifuged at 350g for five minutes, then resuspended in PBS for immunisation. Fusion and Maintenance of Hybridomas Two (CBA x BALB/c)F-| mice were immunised 20 days (i.v.) and three days (s.c.) prior to fusion, with 5x10° tumour cells/ml in PBS. On the day of fusion the spleens were removed, processed and fused with the NS-1 myeloma in a manner which wil l be well-known to persons ski lled in the art. After screening for antibody production the hybrids were cloned by the l imiting di l ution techni que. The hybridoma selected for study in this report was also grown in (CBA x BALB/c) F-j mice injected previously with Freund's adjuvant, from which ascites and/or serum was collected. Serological Methods The sheep anti-mouse i mmu nog! obul i n (SAMG) rosetting assay described in The Journal of Imm unol ogi cal Methods 1978 Vol 20 at pages 173 to 183 was performed in 96 wel l microtiter plates (Disposable Products, Adelaide, Australia) by mi xi ng 25ul target cel ls (5x10°/ml ) with culture supernatants or dilutions of antisera (25ul) for 30 minutes at 4°C, by washi ng three times i n L-15-0.5% BSA and then by adding 25ul of SRBC coated with SAMG using chromic chloride. The SRBC- SAMG -target cell suspension was sedimented by centri f ugation at 200g for three minutes and the percentage of rosettes formed counted after 30 minutes at 4°C. Preparation of Lymphocytes Heparinised blood was collected and centrifuged at 350g for 10 minutes, and the platelets removed. Then 1ml of OMPI • Ficoll Paque (Pharmacia, Uppsala, Sweden) was placed under 6mls of blood; centri f ugation of the suspension at 750g for 10 minutes left a lymphocyte enriched interface which was separate from the red blood cells and gran ul ocytes. These were collected, washed three times and resuspended in LI 5—5% BSA. Tissue Sections Normal and neoplastic tissues were obtained immediately after surgery, snap frozen in a liquid ni trogen-isopentane (BDH Chemicals) slurry, and stored at -70°C. Tissue sections (6um) were cut in a cryostat and placed on glass slides previously washed fo two hours in 90% alcohol. Sections (5um) were also cut from formalin-f xed, paraffin embedded blocks and ..dried onto glass slides. The slides were rehydrated in xylene and alcohol, ready for the immunoperoxidase and immunof 1 uorescence assays. Immunoperoxidase Staining After rehydration formalin-f xed tissue sections were treated with 0.5% H2O2 in PBS for 30 minutes to remove endogenous peroxidase activity, then washed in PBS for 15 minutes (this step was omitted for fresh cryostat sections). Tissue sections were covered with 1:10 dilution of supernatant containing antibody incubated in a humidified atmosphere for 40 minutes at room temperature, then washed for 15 minutes in PBS and 0.2% gelatin. An ammonium sulphate precipitate of rabbit anti-mouse i m munog lobu 1 i n (previously absorbed with fresh human spleen to remove non- specific antibody binding) diluted 1:100 was then applied- and incubated for 20 minutes. The slides were washed for 15 minutes in PBS and 0.2% gelatin, and then covered with a 1:20 dilution of swine anti-rabbit i mmunoglobul in antiserum con ugated wi th horse-radish peroxidase (DAK0- Immunog.lobul ins, Copenhagen, Denmark) for 20 minutes. After the slides were washed in PBS and 0.2% gel atin, di aminobenzi di ne (DAB; 1.5mg/ml, Sigma Chemical Co.) was added for 4-5 minutes. The excess DAB was removed and the slides washed in running tap water for 5 minutes,- counter stained with haematoxylin and then mounted. A monoclonal - 9 - anti body directed agai nst the Ly-2.1 antigen of the moiffre lymphocyte was used as a non-reactive control and a medium control was also included for each experiment. Im unofluorescence Ti s s u e s ect i on s e re s ta i n ed by i n d i ect i munofl uorescence. Sections were i ncubated with a 1:10 dilution of the hybridoma culture supernatant for 40 minutes in a humidified atmosphere at room temperature. The sections were subsequently washed in PBS, and stained with a sheep anti-mouse IgG antiserum conjugated with fluorescein. Stained sections were examined with a Leitz Orthoplan Fluorescene microscope by transmitted darkfield illumination with a narrow band blue excitation. Immunoglobul i n Assays The i mmunoglobul i n isotype of the monoclonal antibody 3E1-2 was determined by Ouchterlony gel diffusion using class specific rabbit antisera to IgM, IgGl, IgG2a, IgG2b (Meloy Lab., Springfield, Va., USA). The anti body titre of 3E1-2 was determined by subjectively examining histological sections (known positive sections) stained by antibody in di lution; the titer being recorded as the next and last issue section which did not stain. Seru _Co _]e£t 0 n _ a_ n d__ Storage Samples of blood were obtained from patients with carcinoma of the breast, and normal female controls. Serum was separated by allowing the blood to clot, then spinning at 750g for 10 minutes. The serum was removed and stored at -70°C until use. idney membrane was used as a source of antigen (as it is eadily available to us than breast tissue), preparations were made by taking 10° cel ls (cell l ines or fresh tissue) and homogenising in 0.25M surcorse (25mM Tris, ImM EDTA, pH7.4) with a Dounce homogeniser at 4°C. The homogenate was spun at 2000g for one minute at 4°C to remove nuclei, and the resulting supernatant spun at 4,200 g for 10 minutes to pel let the crude membrane preparation and the portein content determined by the method described in 'Analytical Biochem stry' 1976 Vol 72 at pages 248 to 254. The assay * §U £4 was performed by adding 50ul of a 200ug/ml stock solution of crude kidney membranes in PBS to a 96 well flexible TV C plate. Membranes were fixed to the plate with 50ul of 0.1% gl utaraldehyde at room temperature for 15 minutes, washed in PBS, then treated with lOOul of 0.1M glycine for 15 minutes at room temperature to block free aldehyde groups. The wells were then washed twice with assay buffer (PBS + 0.5% BSA), then left in lOOul of buffer for 30 minutes at room temperature. Patients' serum (50ul) was added at the appropriate dilution, and to this 50ul of antibody 3E1-2 was added. After 45 minutes incubation at 37°C, the wells were washed three times with assay buffer, then incubated with 10 5 counts per minute/wel l of 125 I RAMG for 45 minutes at 37°C. Plates were washed four times with assay buffer, cut, and counted in a gamma counter. Results Details of Cell Fusion and Screening After cel l fusion, hyb ids were seeded into 384 microtiter wells and growth was observed in approximately 70%. The supernatants were screened by the rosetting assay on the lumphocytes and graπulocytes of two normal individulas, to eliminate unwanted antibodies, then tested by the immunoperoxidase technique on formalin-fixed paraffin embedded sections of the immunising tumour. Several were selected for further characterisation on formal in fixed, paraffin embedded sections of normal breast and other breast tumours and one antibody, 3E1-2, is described herein. 3E1-2 was found to be of the IgM class and usi ng the immunoperoxidase method on tissue sections had a titre on breast carci noma cel ls of 1/2000 for supernatant and 1/20,000 for ascites. Reactivity of 3E1-2 with Breast Carcinoma The anti gen detected by the 3E 1-2 a nt i body survived formal in-f xation and paraffin embedding, and so a large number of carcinomas of the breast, and normal tissues could be easily examined. Of the 37 carcinomas of the breast examined, al l were pos tive for 3E1-2 (see Table 1). Various types of breast carcinoma were represented and OMPI -. included 23 ductal carcinomas, 4 adenocarcinomas, 4 comedocarcinomas, 1 lobular carcinoma and 5 metasta tTc carcinomas. Of the 37 specimens examined, 31 had between 60-100% of mal ignant cel ls staini ng, 5 had 10-60% of malignant cel ls staining and one showed less than 10% of malignant cells staining. Although each histological type stained, the distribution of staining varied. Ductal carcinomas showed staining of both membranes and cytoplasm, whereas a de no ca re i n o a exhibited p edominantl lumi nal membrane staining and little cytoplasmic staining. Comedo (in sutu) carcinoma showed staining of luminal membranes, as well as cytoplasmic staining, while lobular carcinoma stained predominatly cytoplasm, and metastatic carcinoma resembled the ductal carcinomas in staining pattern. In the ^majority of cases (>85%) the staining intensity of carcinoma of breast was either very strong or strong. Thus al l carinomas were clearly positive, although the number of cells staining, and the distribution and intensity of staining varied with the individual tumours. Reaction with Normal, Hyperplasic, Lactating and Benign Breast Tissue To further characterise the tissue specificity of the 3E1-2 antibody, normal, benign, lactating and hyperplastic breast tissue was examined (Table 2). The immunoperoxidase technique revealed that 3E1-2 recognises an antigen present on normal breast tissue. However, it was restricted to only 60% of normal individuals tested, and on average, to 50% of ducts and acini in any one section.- The staining was always of the liminal membrane surface, with the apical cytoplasm s'taining in some cases. This is in contrast to the pattern of staining seen in carcinoma of breast, where staining was cytoplasmic as wel l as membranous, with most sections showing greater than 80% of cells staining. Benign lesions of the breast such as papi l loma, g n aecom as t i a and fibroadenoma showed almost exclusively staining at the luminal membrane surface of neoplastic epithelial cells, a pattern simi lar to that of normal breast. Cystic hyperplasia showed a staining pattern similar to the benign conditions. Lobular hyperplasia however exhibited not only luminal membrane staining but also a staining of tTie epithelial cell cytoplasm, and of the myoepi the! i al cells. Of interest was the finding that a section of prelactating hyperplasia showed no staining of any hyperplastic components, whereas adjacent normal ducts in the same sections showed intense luminal membrane staining. Sections of lactating breast exhibited variable staining, the pattern of staining of this condition is unclear. Thus normal breast tissue and benign conditions of the breast was also positive with the 3 El —2 antibody, but the d i s tr ibut ion of staining was quite different from that seen with breast cancer. Reaction with Normal Human Tissues Monoclonal antibody 3E1-2 showed a rest icted reactivity with normal human tissues (Table 3). In the kidney strong membrane staining was observed in >95% of the distal convoluted tubule cells, but only 20% of those also gave strong cytoplasmic staining. Some collecting ducts exhibited luminal membrane staining, with weak cytoplasmic staining, whereas others did. not react at all. Other significant reactions were only seen in the sweat glands of the skin, the epithelium of the urinary bladder and the su bp opul atio n of lung alevolar cel ls. In the sweat glands the staining was restricted to the canal iculi between the cuboidal epithelial cells. The alveolar cells of the lung show membrane staining of the surface which faces the alveoli; the surface resting on th_e basement membrane did not stain. A moderate degree of staining was seen in the striated ducts of the salivary gland, the myocardium of the heart and the white matter of the brain and spinal cord. Weak staining (possibly background) was seen in the epithelium of the thyroid, stomach and ileum. Tissues showing no staining were liver, prostate, ovary, spleen, colon, adrenal gland, parathyroid gland, bone " marrow, gall bladder, cervix pancreas, lymph node, ileum, stomach, trachea, salivary gland, heart, lung, kidney, skin and thyroid. ' SUREAC P x e_ac_tiy_j_ty__of_3E1-2 with other Human Tumours Monoclonal antibody 3E1-2 was tested on a ranqe of non- breast tumours to further determine the distribution of the "antiqen" detected by the 3E1-2 anti body (Table 4). Using the immunoperoxidase technique it was found that 3 E 1 — 2 recognises an antigen present on renal, endometrial and cervical carcinomas. Staining of malignant cells was both cytoplasmic and membranous and was of similar intensity to that seen on carcinoma of the breast. Lung carcinomas in the main appeared to be negati ve, but i n some, a smal l proportion of cells in between the larger tumour masses were positive. It is probable that these are* alveolar cells, as they constitute 10-30% of cells present, and are always present between l arge groups of non staining malignant cells. The fact that metastatic l ung carcinoma shows no staining supports this belief. Examples of carci noma of colon, malignant melanoma, nasopharyngeal carcinoma, basal cell carci noma (skin), transitional carci noma (urinary bladder), mucinous cystadenoma (ovary) and giant cell tumour of bone were found to be non-reactive with the 3E1-2 antibody. Reactivity with in vitro cell lines An extensive range of in vitro cell lines were examined with the 3 El —2 anti body (Table 5). The antibody fai led to react with 10/10 breast carcinoma cell lines, with 5/5 colon carcinoma cell l ines, or to any other in vitro cel l li ne, including cell s deri ed from melanoma, and tumours of the uterus, lung, p-ancreas, kidney, parathyroi d, and fibroblastic or haemopoietic derived cell lines. As many of these cel l l ines were adherent, and trypsi n was used for their preparation which conceivably could destroy a trypsin sensitive antigen, several of these cell lines were prepared by using EDTA; they sti ll did not react with 3E1 -2. In addition, histologic sections of breast carcinoma did not loose their ability to bind 3E1-2 when treated with varying concent ations of trypsin for up to 30 mi nutes at 37°C. Using the immunoperoxidase technique on air dried monolayers of in vitro cel l l ines have indicated that the 3E1-2 I antibody reacts with cytoplasmic components of a few carcinoma of breast cell lines. Of those cell lines tesfcβ'd (T47D, BT-20, PMC-42, MCF-7 - derived from carcinoma of the breast, PMC-115, PMC-116 - fibroblastic origin), only two T47D and BT-20 reacted with the 3E1-2 antibody. Staining of T47D was restricted to the inner cel l membrane of 5% of cells, while 95% of BT-20 cells gave strong cytoplasmic staining. Molecular Weight Estima ion After cell surface labelling of fresh carcinoma of breast and normal kidney cells by both the lactoperoxidase and chloramine T methods, no specific bands we e found on either 7.5% or 12.5% (reduced and unreduced) SDS-PAGE gels. Appropriate positive and negative control antibodies were used for all gels, as were molecular weight markers. In addition internal labelling of carcinoma of breast cell line BT-20 (which is membrane negative, cytoplasmic positive) with methionine produced no specific bands on either 7.5% or 12.5% non reduced SDS-PAGE gels. Serum Inhibition Study To determine whether 3E1-2 could be used for diagnosis, based on the detection of antigen in serum, a preliminary study was performed on twenty patients with carcinoma of breast. The serum from these patients was used to inhib t the binding of 3E1-2 to a membrane preparation. Serum from normal females (aged 35-65 years) showed a sjnall degree of inhibition, implying that the "3E1-2 antigen" is normally present in serum, but only in -small amounts (see Figs. 1 and Z). By contrast, serum from the patients with primary carcinoma of breast and those with metastases showed significant inhibition of 3 El —2 binding. Fig. 2 shows diagramatical ly an example of this inhibition, comparing it to serum from a normal female control (± standard deviation). These results were quantitated by taking the inhibition graph of each patient, and calculating the "inhibition units" (I.U.) per ml of serum (calculated as the reciprocal of the serum dilution giving 50% inhibition of the maximum binding of 3E1-2 to the membrane preparation 1ϊUR£4 _ OMPI x20). Serum from the patients with localised carcinoma of breast showed a significantly greater degree of inhibitioa, compared to aged matched female controls (p<0.005). Thi s was also the case with patients having a primary tumour and metastases (p<0.01). Serum from patients with etastases inhibited almost twice as much as serum from patients with a local ised lesion only. These two groups were also significantly different from each other (p<0.05). Discussion The production of the murine monoclonal antibody 3E1-2 and its extensive characterisation by the immunoperoxidase technique on normal and neoplastic tissue is descri bed above. The antigen recognised by 3E1-2 survived formalin fixation and paraffin embedding, (a property selected for in the initial screening), and was found to be present on al l carcinomas of the breast and most benign proliferations of the breast although in a different form. However only 60% of normal breast tissue reacted with the antibody, and the majority of normal human tissues were non-reactive. Using a surface membrane assay, no reaction was found with 43/43 in vitro cel l lines, including 10/10 carcinoma of the breast cell l ines. However 3E1-2 has subsequently been found to localise to the cytoplasm of two breast carci noma derived cell lines. An important finding was that 3E1-2 detects an antigen present in serum, which is significantly raised in patients with carcinoma of the breast. In a new approach initial screening was performed on formalin fixed tissue, which led to the identification of an antigen which is resistant to formalin fixation and paraffin embedding. Indeed, thi s antigen al so resi sts fixation by periodate-lysi ne-paraformaldehyde and gl utaraldehyde, and as s uc h, l en d s i tse l f to con ve nt i o na l s t udy by immunohi stochem i cal means without having to resort to using fresh frozen tissue sections. In spite of this the nature of the epitope recognised by 3E1-2 is unknown, as is the molecule it resides on. Surface protein labell i ng experiments using both the 1 actoperoxi dase and chlora ine T methods, on known positive tissues, produced no specific OMPI - - bands by SDS-PAGE analysis. The explanation for this could be that the molecule is not protein and therefore will not label with '"I, alternatively it could be glycoprotein but containing only a few exposed tyros ine residues, and therefore does not label sufficiently to be seen on the autoradiograph. To further study this internal labelling of the in vitro cell line BT-20 with S^ 5 methionine was performed, but again no specific bands on SDS-PAGE gels were obtained. It could be that methionine is present in limiting amounts, but it is not unlikely that it is glycolipid rather than protein particularly as cell membrane glycolipid molecules have been described elsewhere using a similar technical approach. Immunohi stochemica 1 studies have revealed that 3E1-2 recognises an antigen with a restricted distribution on normal human tissue. Indeed, 3E1-2 reacted predominantly with a subpopulation of epithelial cells. This phenomenon of antibodies recognizing epithelial "subsets" is not uncommon and has been previously reported for other monoclonal antibodies to human tumours. The antigen is present on the surface membrane of breast, kidney and lung epithelium, and also on the membrane and cytoplasm of carcinomas derived from these tissue types. All of these tissues are involved in specialised secretory functions and one could postulate a role for the 3E1-2 molecule identified in that process. Compared to other monoclonal antibodies produced to carcinoma of the breast, 3E1-2 reacts with very few normal human tissues. However, comparing antibodies is difficult, as few studies have fully characterised their monoclonal antibodies using sensitive immunohistochem ical techniques - and this is where the ability to test a collection of fomalin fixed tissues is so important. Interestingly, on normal breast tissue and on distal convoluted tubule of kidney, the "3E1-2 antigen" shows heterogeneity of staining, i.e. morphologically identical cells, located adjacent to each other, show considerable variation in staining pattern. This could indicate that the molecule identified may change its expression during the cell cycle, or i n response to changing levels of hormones, chemicals or other substanc___ε required by the cel l or i ndeed, that hi stological y identical cel l s have a different function. Such an hypothesis may explain the failure of 3E1-2 to react with surface components of in vitro cell lines which usually are grown in the absence of hormones and other substances normally present in ' vivo. Detailed examination of sections of breast, including normal, benign, hyperplastic and mal ignant tissues, have been informati ve in that each has shown the expression of the "3E1-2 antigen" during various stages of development, indicating that 3E1-2 defines an antigen important in identifying the different stages of development from normal to malignancy. For example, in normal breast, f i broadenoma, papi lloma and gynaecomastia, the antigen was localised 'to the luminal membrane surface and apical cytoplasm of the epithelial cells. Hyperplastic conditions showed two types of staining. Cystic hyperplasia, a "benign" change of the breast l imited to fibrosi s, sclerosi ng adenosis and cyst formation, shows luminal membrane and apical cytoplasmic staining, similar to that of normal and benign breast conditions. Secondly, lobular hyperpl asia which i s essentially an epithel iosis, showed not only lumi nal membrane staining but al so cytoplas ic staining, and stain'ing of myoepi thel i al cells. This finding is relevant in that only in the case of lobular hyperplasia is there evidence for transition to mal i gnancy and carci noma formation. Few carcinomas develop from fi broadenoma, gynaecomastia and fi brocystic di sease. Our results show clearly that 3E1-2 is localised to both the membrane and cytoplasm of malignant cells (Table 1). In this respect the 3Ε1-2 antibody confirms the link between lobular hyperplasia and carcinoma of the breast, and may be important in studying the genesis of carcinoma of the breast. 3E1-2 does not appear to recognise known components of human mi l k s uc h as cas ie n and lactal bumi n, as immunoperoxidase staining of milk in histological sections "βU E A O PI was negative. The distribution of immunoperoxidase staining and failure of SDS-PAGE analysis of breast tissue discounts hormone receptors for estrogen, progesterone and prolactin as the target. In addition to this the antigen detected has a unique reaction on in vitro cel l lines and tissue sections, when compared to a large range of carcinoma of the breast antigens described ' in the literature. There does however, appear to be some similarity between 3E1-2 and the LICR-LON M8 and F36/22 monoclonal antibodies but in our hands M8 and 3E1-2 have different tissue distributions and F36/22 reacts with the surface membrane of many other different cell lines. Thus 3E1-2 is a "new" breast carcinoma antigen previously undefined. Potentially the most important finding of this study is that the antibody 3E1-2 detects an antigen present in the serum of patients with carcinoma of the breast patients. The results show (see Figs. 1 and 2) that sera from patients with a localised tumour gave significantly more inhibition of 3E1-2, compared to normal age matched female controls. The same was true for patients with metastases. This compares favourably with all other carcinoma of the breast serum markers described to date. The value of the 3E1-2 marker can only be assessed when compared to other markers of breast cancer, used thus for such as CEA and the recently described anti-human MFGM. When this comparison is made, elevated levels of the 3E1-2 antigen occur in most (>85%) of patients with primary breast cancer and metastases (p<0.05). However, with CEA testing elevated levels occur in only 15% of patients with primary tumours and 65% of patients with metastases. For anti human MFGM these values are only 25% and 75% respectively. Although this work is at a preliminary stage the results were significant (p<0.05). The current test, a radioi mmunoassay, can be performed rapidly on large numbers of serum samples and is highly reproducible. In an alternative embodiment of the invention, the 3E1- 2 antibody is used to localise carcinoma of the breast in vivo. This embodiment wil l now be described in greater detail. OMPI . , W1PO Methods Antibodies The monoclonal anti-breast antibody used was 3 E 1 — 2 (IgM), which reacts strongly with membrane and cytoplasm of breast carcinomas and with the luminal membrane of normal breast. Control monoclonal antibodies to murine antigens Thy-1.2 (IgM) and Ly-1.1 (IgG 11) were also used, and in several cases, the patients own IgG was isolated using protein A - affinity chrom atography; an anti-colon carcinoma monoclonal antibody was also used: none of these control reagents reacted by the immunoperoxidase method with normal or malignant breast tissue. The IgM antibodies were isolated from ascites fluid by dialysis against distilled water at 4°C after which the precipitate was collected; IgG antibodies by NH^SO,^^ precipitation. Antibody activity measured either by the rosetting technique referred to above, or by the immunoperoxidase method on sections of breast carcinoma. The purified antibodies were retested for activity after all procedures, filtered through a 0.22um filter (Gelman Acrodisc, Michigan, USA), and tested for pyrogens,. and sterility prior to use. Radio Iodination Purified monoclonal antibodies (lOOg) were labelled with ° I using chloramine T, and the I labelled antibody separated from free iodine on a PD-10 column (Pharmacia Chemicals - Sweden). Control antibodies or human IgG were labelled with '25j b the same method. Patients All patients (except for one with lymphoma) had proven breast carcinoma, and their clinical details are shown in Table 6. One hour before the subcutaneous (sc.) injection of monoclonal antibody each patient received potassium iodide (5 ml at 16.2% w/v), sodium perchlorate (400mg) orally and an intravenous injection of Hyd rocort i s on e (lOOmg) and promethazine (25mg). Radio Imaging Approximately 24 hours after the injection of the radiolabel led antibodies the patients were scanned using a "BUREA OMPI Toshiba GC 402 A gamma camera and a high energy parallel hole col l i ator; computeri zed acquisition using .___an Informatek Simis 4 computer was performed. A window setttng of 50 KeV with a 50% window was used for '25j SC ans and 360 KeV with 20% window for ' 3 'I scans. There was 15% down scatter of 131 I photons into the 125 I channel but since 12 I counts were three times greater than those from 131 I, this amounted to only 10% of counts. Blood pool subtraction was not performed because of the superficial site of the gland. In three patients comparison of . the uptake of specific antibody and non-specific i mmu nog! obul in control in the lymph nodes was performed by using l I-3E1-2 (specific antibody) and as control 125 I-Thy-1.2 (IgM); 125 I-Ly-1.1 (IgG), 125 I-30.6 and 125 I-homol ogou s IgG. The fraction of radiolabel led antibody localising in the axillary nodes (F) was estimated by measuring the nodal uptake (N) using the alpha camera, corrected for "down scatter" from the ' " ' I channel and comparing this to the amount of radi ol abel led antibody found at the time of the imaging (Im) and time of injection (In) so that F = N xA, where A = the correction factor (2.5) used for lt3 I attentuation in the axil la, determined by placing a source bottle under the patients armpit and comparing this to the actual radioactivity in the bottle (ie 400 vs 1000 cpm). Results In this embodiment patients were injected w ith radiolabel led 3E1.2 antibody by the subcutaneous route into the dig-ital web space. The rationale of this study was that the antibody would enter the lymphatics and pass through draining lymph nodes and detect metastatic tumour residing there. The results are shown in summary in Table 6. Discussion The currently available techniques for the detection of breast cancer include pal pation, x-ray mammography, thermogr ap hy, ultrasound, di aphr anog raphy and nuclear magnetic resonance. However, these methods distinguish between benign and malignant lesions with difficulty. It is considered to be important to be able to detect metastases ■" BUREA OMPI in drai ni ng axi 11 ary lymph nodes in carcinoma of the breast as this serves to distinguish the early and late stages^f the disease and currently is used to design appropria-te therapy. With this in mind we have attempted to detect small metastases in the draining axillary lymph nodes of patients with breast cancer. The method of using radiolabelled monoclonal antibodies continued with scintography has recently been receiving some attention for the detection of a variety of tumours. The re-emergence of this technique - attempted unsuccessfully some years ago - has been developed because of the advent of monoclonal antibodies with at least some degree of specificity fo tumours. Thus using the intraveneous route we and others have been able to detect both primary and secondary tumour arising from a range of different tumours. At this time it is not clear whether very small tumours can be detected and there is clearly a need for the refinement in the use and production of these monoclonal antibodies, the type of radi ol abel 1 i ng and the site and route of injection. The antibody used herein, 3E1.2 was used with little success by the intraveneous route to delineate secondary deposits from breast cancer. The results obtained (data not shown) were similar to those described using other antibodies. However it appeared that small tumours would evade detection by intravenous means - the major problem being the high content of circulating radiolabelled antibody which, after subtraction, leaves little detected in the tumour. To overcome these problems and to determine whether subcutaneous antibody administration was a better route to the draining lymph node via the lymphatics, we used radiolabelled antibody given subcutaneously. In the patients studied we were clearly able to detect metastases and to confirm their presence in those patients who, by other means were already suspected to have tumour involving axillary lymph nodes. Thus the method demonstrates that radiolabelled antibody can react with tumour in draining lymph nodes. Of greater importance however was the finding of additional lymph nodes which had not been clinically - 22 - palpable and those whose presence was not suspected i.e. IJie immunoscintography test was more sensitive than convention-al clinical examina on- for the detection of metastases in draining nodes. Whether this technique is more sensitive than conventional 1 ymph angi og raphy remains to be seen. None -t he-less, this is one of the first examples where monoclonal antibodies appear to be superior to the existing commonly used cl inical method for the detection of small tumour deposits. Several comments should be made regarding the method. Firstly, it did not appear necessary to use subtraction with a non-specific antibody as the specifically labelled antibody was retained in the abnormal lymph node but rapidly cleared from the normal lymphatics and normal nodes. Secondly, the kinetics of the disappearance of antibody were such that scanning between 16-24 hours appeared to be more suitable than earlier or later scans. Thirdly, it was clear thai "pathological" lymph nodes retained radiolabelled antibody both specifically (when involved with carcinoma of the breast) and non-specif ical ly (when involved with other diseases such as lymphoma). This is not unexpected because of the greater vascularity of involved lymph nodes and because of the possibility of some hold up in lymphatics draining in these nodes. In this context, is the immunoscintography described herein any different than lymphangiography using more conventional reagents. We feel the method does have advantages over the conventional lymphaπgiogram in that normal lymph nodes are not detected; there is twice as much (or more) specific antibody detected, compared with non-specific and in a grossly abnormal lymph node the collection of antibody was not greater than that found in other tissues. However the sensitivity of the method needs improvement, for example rad io iodi nated antibody is not the best method for labelling because of the short half life in vivo of such antibodies. However it should be noted that in recent studies in both mice and guinea pig, tumours of 2.3mg could be detected and there is no reason why such a sensitivity should not be obtained in the type of studies described herein. However, whether the method will approach that to detect micro - etastases in boαg marrow wil l depend on the amount of antibody given, th-e qual ity and quantity of the radiolabel, and the detection system. Possibly the combined use of several antibodies to different epitopes on the cel l surface may lead to a magnification and greater sensitivity. However, the essential question to ask is whether this procedure - both simple and painless to perform - represents a real advance in the early diagnosis of breast cancer. We believe an advance has been made since no other technique is currently avai lable which can so readi ly disti nguish between normal and malignant nodes. We have therefore shown that (i) palpable nodes can be detected by immunoscintography, (ii) normal nodes do not take up the antibody, (ii i) abnormal nodes (lymphoma) only marginal ly retain antibody, (iv) localisation of the anti body is specific by at least a factor of two, (v) nodes not clinical ly palpable can be detected, (vi) the subcutaneous site of injection is superior to the intravenous route for this antibody. In the few patients studied, palpable lymph nodes were readi ly detected but impalpable nodes were also found. What is now required is a l arger prospecti ve study where patients, considered cl inical ly to be stage 1 (carci noma localised to breast only) are subjected to immunosci ntography prior to operation, and the immunoscintography findings compared with subsequent hi stologi cal-exam i nation of axillary lymph nodes. At thi s time there are a number of patients who clinically appear to be stage 1, but at operation are found to be stage 2 or stage 3. If this technique alters the management of these patients and reduces the extent or necessity for mastectomy, combined with an earl ier diagnosis, the technique can then be pronounced to be of use. Z Table 1 Reaction of monoclonal antibody 3E1-2 with primary and .metastatic carcinomas o the breast 3 Tumour Type No. positive % mal gnant cells Range of Staining Intensity c No.tested cells staining 0 Membranous Cytoplasmic Min. Max. Ductal Carcinoma Well differentiated 5/5 20 100 ++++ - 0 ++++ - o Moderately " 13/13 25 100 ++++ - 0 ++++ - + Poorly " 5/5 80 100 ++++ - 0 ++++ - ' + Adenocarcinoma 4/4 25 100 ++++ - 0 ++ - + Comedo/Mucus 4/4 95 ++++ - 0 +++ - 0 Lobular 1/1 100 100 - 0 +++ - ++ Metastatic 5/5 40 100 ++++ - + ++++ - 0 Tissues were tested by immunoperoxidase and graded: 0 = negative, + » weak, ++ = moderate, +++ = strong, ++++ = very strong Indicates the minimum and maximum amount of malignant cells staining (%) fo a particular tumour type, from a number of different individuals- Indicates the range of cytoplasmic and membrane staining intensity for a particular tumour type from a number of different individuals. ISURE OMPI Ϊt5 Table 2 Reaction of monoclonal antibody 3E1-2 with normal, benign, lactating and hyperplastic breast tissue by the immunoperoxidase technique 3 Tissue No. staining Reaction No. tested Resting Breast Acini Epithelial cell 2/5 +++-++ Luminal membrane 3/5 ++++-Q Myoepithelial cell 1/5 +++_++ Secretion 1/5 +++ Intralόbular ducts - Epithelial cell 1/5 +++-++ Luminal membrane 3/5 ++++-0 Myoepithelial cell 1/5 +++-++ Secretion 1/5 +++ interlobύlar ducts Epithelial cell 1/5 +++-++ Luminal membrane 3/5 ++++-0 Myoepithelial cell 1/5 +++-++ Secretion 1/5 +++ Lactating Breast Acini Epithelial cell 1/2 +++-++ Luminal membrane 1/2 +++-0 Milk Fat Globule Mem. 1/2 +++-0 Myoepithelial cell 1/2 ++ Secretion 0/2 0 Gynaecomastia - - Ducts Luminal membrane 5/5 ++++-0 Epithelial cells 5/5 +++-0 Myoepithelial cells 3/5 +++-0 Secretion 4/5 ++++-+++ Pre Lactating Hyperplasia Acini Epithelial cells 0/1 - Luminal membrane 0/1 - MFGM 0/1 - Myoepithelial cell 0/1 - Secretion 0/1 - Lobular Hyperplasia Acini Luminal membrane 4/4 ++++-0 Epithelial cell 4/4 ++++-0 Myoepithelial cell 4/4 ++++-0 Secretion 0/4 Cystic Hyperplasia Cystic Ducts Luminal membranes 2/2 ++++-0 Epithelial cell 1/2 +++-0 Myoepithelial cell 0/2 Cystic flui.d 0/2 Fibroadenoma Ducts Luminal membrane 4/5 ++++-0 Epithelial cells 4/5 +++-0 Myoepithelial cells 0/5 Secretion 2/5 +++-0 Papilloma . Luminal membrane 1/1 ++++-0 Epithelial calls 1/1 ++-0 Myoepithelial cells 0/1 Secretion 0/1 a Tissues were tested by the immunoperoxidase technique and graded 0 = negative, + = weak, ++ = moderate, +++ = strong, ++++ = vveerryy sstrong. ' Table 3 Reaction of monoclonal antibody.3E1-2 with normal human tissues by the immunoperoxidase technique 3 Organ/Tissue Cell Type Reaction * Skin Keratin Stratum Granulosum Stratum Spinosum Stratum Germinatium Sweat Glands +++ Sweat Ducts Sebaceous Glands Trachea Surface Epithelium Submucosal Glands Lamina Propria Lung Pneumocyte type I Pneumocyte type II Bronchiole Teritary Bronchus Primary Bronchus * Spleen Capsule. White pulp -Red pulp Lymph Node Capsule Cortex Medulla Thyroid Epithelium • "C" cells Colloid Parathyroid Chief cells Oxyphil cells Adrenal Gland Zona Glomerulosa Zona Fasiculata Zona Reticularis Medul1a Kidney Distal Convoluted Tubule ++++ Proximal Convoluted Tubule Glomeruli Loop of Henele Collecting Duct +++ Vasa Recta Urinary Bladder Transitional Epithelium +++ Smooth Muscle Salivary Gland Mucinous Acini Myoepithelial Cells Striated Ducts Intercolated Duct Serous Acini Serous Demilune Stomach Secretory Epithelium Non secretory Epithalium Parietal Cells Zymogenic Cells Colon Surface Epithelium Crypt Epithelium Sub Mucosa Ileum Surface Epithelium Crypt Epftheliu Sub Mucosa Liver Paranchyma Portal Tri ad Kupffer Cel l s Gal l Bl adder Col umnar Epithel ium Sub Mucosa Pancreas Acinar Cells Centro Acinar Cells Exocrine Ducts Islets of Langerhans Prostate Glands " Ducts Stroma Ovary Follicles Stroma -BURE OMPI Cervix Endocervix Stratified Squamous Epithelium Lamina Propria Heart Epicardium Myocardium ++ Pericardium Brain Grey Matter White Matter ++ Spinal Cord Meninges White Matter ++ Grey Matter Bone Compact Bone Cancellous Bone Bone Marrow Hyaline Cartila* Muscle Smooth Skeletal Cardiac ++ Connective Fibroblasts Tissue Mast Cells Macrophages Blood " • " Lymphocytes Monocytes Eosinophils Polymorphs Platelets 3 Tissues were tested by immunoperoxidase and graded: + - = negative, + = weak, ++ = moderate, +++ = strong, ++++ = very strong These tissues were also tested by immunofluorescence These tissues were also tested by Fluorescent-Activated Cell Sorter (FACS) OMPI ™ Table 4 Reaction of monoclonal antibody 3E1-2 on the other human tumours 3 No. stained Reaction % malignant No. tested Lung carcinoma Squamous cell carcinoma 5/7 ++++ 20% Adenocarcinoma 1/1 ++++ 30% Alveol r carcinoma 1/1 ++++ 10% Large Cell carcinoma 0/1 - - Small Cell carcinoma 0/1 - - Secondary carcinoma 0/4 - - Colon carcinoma 0/5 - - Renal carcinoma 3/4 ++++ 70% Endometrial carcinoma 4/4 ++++ 60% Cervical carcinoma 4/4 ++++ 30% Malignant melanoma 0/5 - - Histiocytic lymphoma 0/2 - - Nasopharyngeal carcinoma 0/1 - - Basal cell carcinoma (skin) 0/1 _ m Transitional cell carcinoma (bladder) 0/1 Mucinous cyst adenoma (ovary) 0/1 Giant cell tumour (bone) 0/1 S O PI - ό - a Tissues were tested by immunoperoxidase and graded: + = weak, ++ = modera^, +++ = strong, ++++ = very strong; % cells reacting accorded subjectively by two investigators. Staining between neoplastic cells, probably pneumocytes (see text) Tabl e 5 Reaction of monoclonal antibody 3E1-2 with surface components of in vitro cel l l ines 3 Cel l s Tested 3E1-2 Cel l s Tested 3E1-2 Breast,carcinoma Colion Carcinoma MCF-7 HT29 T 7D Colo 206 BT-20 Colo 394 DA-MB-231 Colo 463 CAMA-1 Colo 440 Maiignant Melanoma PMC 42 LiBr H50578T RPMI 8372 HBL-100 (Benign) RPMI 7932 Colo 533 Colo 239F Colo 525 Ovarian Carcinoma Colo 316 Kidney Carcinoma Colo 319 Colo 293 Colo 526 ^ (uterus) Parathyroid Carcinoma Pancreatic Carcinoma PT-1 Colo 357 Foetal Tissues Haemopoietic Lung U266B Skin Bordln HMy-2 Fibroblasts MOLT-4 ~ P C-115 CCRF-HSB-2 PMC-116 PEER CEM .. . DAUDI OZ-B KM-3 ARH-77 HS-SULTAN RAJI U937 Tissues were tested by SA G-RFC and the reaction graded as +«occurred; ++=strong; ±=weak; —absent __O PI TABLE 6 SUMMARY OF PATIENTS AND DETAILS PATIENT HISTORY CLINICAL DETAILS RADIQIMAGING COMMENT PRIOR TO SCANNING RESULT 83 yrs. primary tumour 1 R breast; node (i) Nodes not visible Scan verifies detected 6 w s prior to palpable in R in iv. study clinical examination, scan receiving Tamoxifen axilla (ii) R axilla (stage III). positi e L axilla negative 73 yrs. (R) radical lymphoedema R arm, No uptake of anti¬ Clinically negative, mastectomy 1978 stage nodes not palpable body in axilla in scan negative. II, superficial nodes two sc. studies. dissected 1979 and irradiated; 1983 tumour noted in L breast. 65 yrs, L breast L axilla node L axilla nodes in Scan verifies clinical mastectomy 1979, palpable 3 sites. R axilla examination, also recurrence 1983, 1984 negative twice. confirmed by needle pleural effusion (stage aspi ation. IV). TABLE 6 (cont) SUMMARY OF PATIENTS AND DETAILS PATIENT HISTORY CLINICAL DETAILS RADIOIMAGING COMMENT PRIOR TO SCΑ " NNTNG " RESULT 37 yrs, radical Nodes not palpable Small nodes detected Nodes not palpable mastectomy L breast in R and L axillae scan positive, 1980 (stage II) 2° in aspiration positive, bone marrow and vertebrae detection of nodal metastases prior to clinical detection. Right mastectomy 1979 R arm lymphoedamatous Uptake in left Scan consistent with (stage II ) sc. tumour deposits, axilla and foci in sc. tumour in R arm L axillary node palpable R arm and L axillary node J Right mastectomy 1976 Mild oedema R arm, L R lymphatic obstruc- Two fold detection (stage I ) 1984 axillary and supra- tion L node seen with of specific antibody Chemotherapy and clavicula nodes specific and less in L node compared recurrence L axi llary palpable with non-specific to non-specific .:j deposi ts antibody antibody. I 68 yrs , 2 month history R axillary and R and L axillae L node detected prior of R breast mal ignancy supraclavicular nodes uptake in nodes. to clinical detection. 1 with supraclavi cular and palpable Non-specific scan Specific to non¬ Λ axi l lary nodes (stage IV) also positive but specific 2:1. less intense ale, non-Hodgkins Gross hepatomegaly Negative Slight retention, no and splenomegaly specific binding of bilateral cervical, antibody. axillary inguinal nodes positi e Left

Claims: 1. A cell line which produces a monoclonal antibody risin a predominant specificity to breast cancer antigens. — 2. A continuous cell li ne which produces breast cance antibodies identified as clone 3E1-2 which produces breas carcinoma antibodies in vitro in hypoxanthi ne-a i nopteri n thymidine medium comprising a fused cel l hybrid of huma aden oc ar ci noma -p ri me d BALB/c mouse sple.en cells, and NS- mouse myeloma cells, said antibodies reacting with a breas tissue antigen. 3. A monoclonal antibody having a predominant specific.it to breast cancer. 4. A method of detecting breast cancer antigens in bloo serum secretions or tissue comprising the step of applyin to a blood serum or secretion to be tested a monoclona antibody having a predominant specificity to breast cancer. 5. The method of Claim 4, wherein said monoclonal antibod is applied, using the i munoperoxi dase technique, t formal in -fixed or fresh neoplastic ti ssue sections, an detecting the presence or absence of showing unifor staining of the cytoplasm and membrane. 6. The method of Claim 4, wherein said monoclonal antibod is applied, using a_j a_d_ 2i_m ^ mu_ a_s_s_a_y_^e_ J n_ _e_, to bloo serum, and detecting the presence or absence of significan inhi bition of monoclonal antibody binding to a membran preparati on. 7. The method of Claim 4, wherein said monoclonal antibod is appl ied, usi ng a.n__E L.I_^A_^e_c_]ιn_ u_e_, to blood serum, an detecting the presence or absence of significant inhibitio of monoclonal antibody binding to a membrane preparation. 8. The method of Claim 4, wherein said monoclonal antibod is radiolabelled and injected intr venously and detectin the presence or absence of axi l l ary lymph nodes b immunoscintography.

Inventors: MCKENZIE IAN FARQUHAR CAMPBELL, MCKENZIE, IAN, FARQUHAR, CAMPBELL
Applicants: UNIV MELBOURNE, THE UNIVERSITY OF MELBOURNE
Application year: 1984
Main classification: C12N15
IPC codes: A61K51, C07K16