FI94291B - Monoclonal antibodies - Google Patents

Description

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Monoclonal antibody

By division separated from application 863375.

5

Human lung cancers cause most cancer deaths in men and are bypassing breast cancer as the most common cause of cancer death among women (Cancer Facts and Figures, 1983). This disease can be divided into four main histo-10 logical types, i. epidermoid (30%), glandular cancer (35%), large cell disorganized (15%) and small cell (20%). Most cases of lung cancer cannot be cured with chemotherapy and radiation therapy. Chemo-therapy and radiation therapy may affect small cell lung cancers by reducing size but not completely curing it. Complete surgical removal of the tumor appears to be the only effective treatment. Unfortunately, however, less than 30% of lung cancer patients have tumors that can be completely removed by surgery, and less than one-third of these potty-20s live 5 years after the apparent complete surgical removal of the tumor. Therefore, there is a great need to provide methods that would allow for earlier detection of lung cancer and better determination of the prevalence of cancer.

25 * Monoclonal antibodies can be used for these purposes. However, a prerequisite is to find antibodies to antigens that are much more present in lung cancer than in normal adult tissues. Given the heterogeneity of tumor cell populations, the presence of multiple determinants in the same antigen molecule, the predicted differences between antigens in terms of suitability as diagnostic markers compared to therapeutic targets, and the different biological properties of different antibodies to the same antigen, a large number of different antibodies may be required.

Human monoclonal antibodies against lung cancer antigens are described in Sikora et al., Br. J. Cancer 2

Q A Q O 1 y t c. s I

(1981) 43.:696-700. Monoclonal antibodies that show specificity for several types of human lung cancer are described in Outitta et al., Proc. Natl. Acad. Sci. U.S. (1981) 78: 4591-4595. Human lung-5 cancer-associated antigens as determined by monoclonal antibodies are described in Varki et al., Cancer Research (1984) 44: 681-687.

Mouse monoclonal antibodies to the glycolipid ganglio-N-10 triosylceramide (asialo GM ^ are described in Young, et al., J. Exp. Med. (1979) 150: 1008. Expression of asialo GM ^ in patients with Hodgins disease, in cells is described in Kniep, et al., J. Immunol. (1983) 131: 1591-1994.

U.S. Patent Application No. 667,521 describes certain monoclonal antibodies against non-small human lung cancer tumors.

Continuous cultures of fused cells secreting antibodies of predetermined specificity have been described by Köhler et al., Nature (1975) 265: 495-497.

The present invention relates to novel monoclonal antibodies having an antigenic binding site that binds to (a) a carbohydrate antigen that is similar to, but not identical to, ganglio-N-triosyl-ceramide, labeled asialo-GM1, and that is non-small cell. lung cancer cell surface determinant; and (b) a protein antigen having a molecular weight of about 20,000 daltons and associated with human non-small cell lung cancer.

Preferably, such antibodies can be produced using a hybridoma with accession number ATCC HB8677.

35

The present invention relates to novel monoclonal antibodies that recognize determinant sites in a glycolipid antigen associated with human non-small cell lung cancer (NSCLC) cells. The term "NSCLC cells" includes epi- 3 Ο Λ O .'Λ Ί

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dermoid cancer cells, glandular cancer cells, and large cell disorganized cancer cells. The determinant site may also be present in antigens of some other cancers, e.g., some breast cancers, and thus the antibodies of the invention will also bind to these other cancer cells. The present monoclonal antibodies bind to a much lesser extent in normal adult cells than in tumor cells. The term "bind to a much lesser extent" means that binding is not detectable by immuno-10 histological methods. Mouse hybridomas secrete monoclonal antibodies.

The monoclonal antibodies of the present invention can be used in methods for determining the presence of a malignant condition in human lung tissue and other human tissues. The method examines the presence in the tissue of a glycolipid antigen having a terminal carbohydrate sequence: GalNAcS1-4GalS1- * 3GalNAcS1- * 4GalS1 and similar antigens such as ganglio-N-triosyl-cerami-20 di, e.g. For example, the tissue may be contacted with an antibody that recognizes a determinant site in a cell associated with a glycolipid antigen that has the aforementioned terminal carbohydrate sequence or a functional equivalent or portion of such an antibody.

One such method involves determining the presence of NSCLC cells in a sample suspected of containing such cells. The sample is contacted with a monoclonal antibody of the invention capable of distinguishing such cells from other cell types that may be present in the sample. The fusion is performed under conditions in which the antibody binds to such cells. After ligation, it is determined whether or not the antibody is bound to the cells of 35 samples. This binding is proportional to the presence or absence of NSCLC cells in the sample. Generally, the sample is contacted with the labeled mono-clonal antibody specific binding partner. This label is capable of producing a detectable signal.

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Another diagnostic method involves locating the tumor using antibodies or antibody particles that have been appropriately labeled (e.g., with a radioisotope) and then injected into patients. This method provides better ways to monitor cancer patients for the extent of the disease and to detect changes brought about by treatment.

The present invention relates to novel antibodies that bind to human NSCLC cell antigen and to certain diagnostic methods using these antibodies. The monoclonal antibodies of the invention can be prepared according to the standard methods of Köhler and Millstein, supra. For example, human lung cancer cells from multiple fluid outbreaks or cultured cells from non-small cell lung cancer or cells from a normal dead lung are used as the immunogen. These cells are injected into the mouse and after a sufficient time the mouse is killed and the spleen cells are harvested. Spleen cell chromosomes encoding the desired immunoglobulins are stored by fusing spleen cells with myeloma cells or lymphoma tumor cells, usually in the presence of polyethylene glycol. The resulting cells containing the fused hybridomas are allowed to grow in a selective medium such as HAT medium, and the surviving cells are grown in such medium using limiting dilution conditions. The cells are grown in a suitable container, e.g., microtiter wells, and the supernatant is screened for monoclonal antibodies of the desired specificity.

30

There are several methods for improving the yields of monoclonal antibodies, such as injecting hybridoma cells into the peritoneal cavity of a mammalian host that accepts the cells and collecting aqueous humor. If an insufficient amount of monoclonal antibody is collected from the aqueous humor, the antibody is collected from the blood of the host. There are several conventional ways to isolate and purify monoclonal antibodies to release monoclonal antibodies 94291 from other proteins and other contaminants (see Köhler and Millstein, supra).

One monoclonal antibody of the present invention is designated L6. It recognizes a cell surface glycolipid antigen that has been identified as typical of human NSCLC cells and certain other human cancer tumor cells. Based on the cross-reactions of several known glycolipids with L6 antibodies and the reactivity of GalNAc-GMj with L6 antibody, it has been concluded that the L6 glycolipid antigen has the following terminal sequence: GalNAcS1- * 4GalSl- * 3GalNAcfil * 4GalS1- * R, where R is a carbohydrate that is still unknown. A particularly novel feature of the above antigen is that it is free of sialic acid residues and to date has not been known to be associated with human tissue. Sialosyl derivatives having the above-mentioned terminal sequence are described in Svennerholm et al., (1973) J. Biol. Chem. 248: 740-742 and Iwamori et al., (1978) 20 J. Biochem. M: 1601. In addition, sialic acid derivatives having the above order have also been identified as blood group -Sd antigens in Donald et al., (1984) Biochem. Soc. Trans. 12: 596-599 and Blanchard et al., (1983) J. Biol. chem. 258: 7691-7695.

The L6 antibody also precipitates the protein antigen from biosynthetically labeled NSCLC cells. This antigen is identified as a band by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) with a molecular weight of about 20,000 daltons. This antibody is of the IgG2a isotype. It does not detectably bind to normal cells such as fibroblasts, endothelial cells, or epithelial cells in major organs. The L6 antibody is produced by the mouse L6 hybridoma.

35

Also within the scope of the invention are useful binding fragments of the above monoclonal antibodies, such as Fab, F (ab ') 2 »Fv fragment, etc. Antibody fragments are obtained by conventional methods. For example, useful binding fragments can be prepared by peptidase lysis of antibodies using papain or pepsin.

Although the above specific example of the novel antibody of the invention is directed to an antibody that binds to specific determinant sites of the respective antigens and belongs to the IgG2 subclass from a mouse source, this is not intended to limit the invention. Above, this antibody and those antibodies that are functionally similar to the above antibody, whether from a murine source, another mammalian source, including a human, or other sources, or combinations thereof, are within the scope of this invention, as are other isotypes. By the term "functional similarity" is meant that the antibody is capable of binding to the determinant site described above and is capable of competing with a particular antibody of the invention for such a site.

Namely, such an antibody, when combined with a sample containing a cell or cell fragment having such a determinant site, binds to such a determinant site and prevents the antibody of the invention from binding to that site. In addition, since the antigen of the invention may have more than one determinant site, the invention includes monoclonal antibodies that recognize determinant sites other than those determinant sites recognized by the aforementioned monoclonal antibody.

The invention includes the diagnostic use of the L6 antigen in humans and the use of similar antigens such as asialo-GalNAc-GM1 and asialo-GM2. The antigen can be purified by conventional methods such as immunoprecipitation as described in Young et al., J. Exp. Med. (1979) 150: 1008-1019.

One method comprises determining a malignant condition in lung tissue and other human tissue by examining the tissue for the presence of a glycolipid antigen having the properties of an L6 antigen or ganglio-N-triosylceramide. The term "malignant condition" refers to growth failure

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94291 7 the presence of cells including in situ cancer cells, neoplastic, malignant or tumor cells or the like. The term "having similar properties" means that the antigen reacts with an antibody that recognizes the L6-an-5 antigen or a similar antigen such as asialo-Gal-NAc-GM ^ or asialo-GM ^. For example, the sample may be contacted or combined with a monoclonal antibody of the invention, such as an L6 antibody or an antibody having similar properties to those described in Young et al., Supra, or Kniep. et al., supra. The fusion is performed under conditions in which the antibody binds to malignant cells. After co-administration, it is detected whether the antibody has bound to malignant cells in the sample, i.e., the immune complex of the antibody and antigen site is not bound to the sample. This formation of an immune complex is proportional to the presence of malignant cells in the sample.

A specific example, illustrative and not limiting, is a method for determining tumor cells from excised tissue. The above method is applied to a sample that is a piece of tumor obtained after tumor removal. The dissected tumor is treated to obtain bodies, which treatment first involves freezing the tumor or tissue, usually freezing immediately after dissection. The frozen tissue layer is then cut into pieces using e.g. a cryostat.

The tumor body obtained as described above is contacted with a monoclonal antibody of the invention, and then another antibody labeled with a detectable label is directed against this monoclonal antibody.

The excised sample, e.g., a tumor body, is contacted with the first monoclonal antibody under conditions in which the antibody binds to malignant cells. Incubation is generally performed in an aqueous medium, such as phosphate buffered saline, 94291 8 containing small amounts of sodium azide, in a suitable container, such as a glass Petri dish, for about 15-30 minutes at a temperature of about 20-30 ° C. The amount of antibody used is generally sufficient to produce detectable binding, i. to produce a detectable amount of immune complexes between the antibody and the determinant or antigenic site in question.

After incubation, the body is washed to reduce or eliminate non-specifically bound antibody and then examined for the above-mentioned complexes formed when the monoclonal antibody binds to cells in the sample that have antigenic sites. Binding is proportional to the presence of malignant cells in the body. Accordingly, binding is determined, for example, by contacting a sample with a labeled monoclonal antibody-specific binding partner. The label is capable of producing a detectable signal and may be a radioactive label, a chromophore such as a fluoro-20 ringing agent, an enzyme or the like.

An example of a technique using the above approach is immunofluorescent staining. In this method, frozen tumor sections are fixed on a glass plate with acetone and incubated with a monoclonal antibody, e.g. in a Petri dish. After washing with an appropriate buffer, such as e.g. phosphate buffered saline block is placed in a petri dish and contacted with the labeled monoclonal speci-30 antibody in the printing binding partner, which may be, for example. The monoclonal antibody specific for the labeled antibody. Since in most cases the monoclonal antibody is obtained from a mouse source, a labeled anti-mouse immunoglobulin specific for the mono-35 clonal antibody can be used. Such immunoglobulins can be produced by conventional methods by injecting a murine antibody into a suitable host, waiting for an appropriate time, and collecting the anti-mouse immunoglobulin from the blood of the injected host.

il 94291 9

After the plate is washed a second time, e.g. with aqueous buffer, the bodies can be covered with a fluorescent antibody fixing liquid and a cover plate and then examined under a fluorescence microscope to determine the binding of the monoclonal antibody to the body. Determining binding may also include identifying such a binding site in the sample.

Binding of a monoclonal antibody to a sample can also be determined using a monoclonal antibody covalently bound to a label capable of producing a detectable signal, such as a radioactive moiety, a chromophore containing dyes and fluorescent agents, or an enzyme. The number of labels to be used per antibody will generally be determined by the requirements of the diagnostic method in which the labeled antibody is used and the sites available to associate the label with the antibody.

Methods for conjugating labels to antibodies and antibody fragments are known in the art. Such methods are disclosed in U.S. Patents 4,220,450, 4,235,869, 3,935,074 and 3,996,345.

Another example of a technique in which a monoclonal antibody of the invention can be used is immunoperoxidase labeling (Sternberger, Immunocytochemistry, John Wiley & Sons, New York, 1979, pp. 104-169, modified by Garriques et al., Int. J. Cancer (1982) 29:511-515). The tissue to be tested is attached as a suitable solvent, such as acetone, to a support, such as a glass plate. The tissue is then incubated with the monoclonal antibody and then washed free of unbound antibody. The tissue is then incubated with rabbit anti-mouse IGG, washed to remove unbound antibody, combined with the mouse peroxidase-antiperoxidase complex, washed to remove unbound conjugate, and then treated with the enzyme substrate. After this treatment, the plate is examined to detect the signal.

94291 10

The antibodies of the invention can be used in a method for determining the presence of a malignant condition, for example, by examining a cell sample detached from the lung, such as sputum, or a cervical preparation. By the term "detachable" 5 is meant that the sample comprises discrete cells or cell clusters obtained by scratching or washing the surface of the tissue, which cells detach as separate or groups or layers. The detachable cell sample should be separated from the excised tissue, such as that obtained by biopsy. Contact with 10 samples and antibody is performed under conditions such that the antibody binds to the antigen site. After co-administration, it is determined whether or not the antibody binds to the antigenic site, and this is proportional to the presence of a malignant condition in the lung.

15

To determine the presence of a malignancy in the lung, a sputum cell sample for use in the method would be obtained from the sputum. The method may be useful in determining a malignant condition of the airways, including oral pharyngeal, oral, etc., in detachable cell samples and from the gastrointestinal tract.

Next, the detachable cell sample is contacted with the above-mentioned antibodies under conditions such that the antibody binds to specific antigenic sites: in the sample to form antigen-antibody complexes. This antigenic site may be present in the cells or cell fragments of the sample. Generally, the sample is placed on a suitable support, such as a slide, usually a glass, or some suitable material. The detachable cell sample is usually applied to the plate to provide a thin layer · from the sample to the surface of the plate. Contact between the sample and the antibody is generally performed in an aqueous buffered medium. Buffers that can be used include phosphate-35 ti, tris, bicarbonate, etc. The pH depends on the nature of the sample and the antibody, and is generally in the range of about 5-8. The aqueous medium may further contain organic polymeric solvents in amounts of about 0-40%. Organic polar

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94291 11 solvents are water soluble and generally have about 1 to 10 carbon atoms and about 1 to 4 oxygen atoms. The antibody is present in the aqueous medium at a concentration of about 1-100 μg / ml, preferably about 10-20 μg / ml. The temperature during contact between the sample and the antibody is generally about 4-40 ° C, preferably about 10-30 ° C. The contact time is generally about 15-120 minutes, preferably about 30-60 minutes.

After the contact time between the sample and the antibody, the support 10 is generally treated to remove unreacted antibody. This is usually done by washing the support with an aqueous, usually buffered medium. In general, the amount of wash solution should be sufficient to remove unreacted antibody.

15

Next, the binding of the antibody to the antigenic sites of the sample is observed, which binding is proportional to the presence of a malignant condition in the sites, i.e., the sample is examined to determine how many antigen-antibody (immune) complexes have formed. It should be noted that in some cases very small amounts of the antigenic sites in question are present in a detachable cell sample. However, in malignant conditions, antigenic sites are present in large numbers and this latter state is easily distinguishable from non-malignant conditions by this method because a large number of antigen-antibody complexes can be measured in the presence of a malignant condition. To determine binding, means are provided in the assay system to provide a detectable signal. For example, the antibody used in the assay can be conjugated in an assay to a label capable of producing a detectable signal. The label may be a radioactive moiety, a chromophore comprising dyes and fluorescent agents, an enzyme, or the like. The number of labels used on the antibody is determined by the requirements of the method and the sites available for binding the label to the antibody.

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Alternatively, the plate may be washed in connection with the labeled specific binding partner for the antibody, which may for example be labeled, the antibody used, the specific antibody. Once the monoclonal antibody is derived from a mouse source, a labeled anti-mouse immunoglobulin specific for the antibody used can be used. Such immunoglobulins can be produced according to conventional methods by injecting a monoclonal antibody into a suitable host, waiting for an appropriate time, and collecting anti-mouse immunoglobulins from the blood of the injected host. When using a labeled specific antibody binding partner, the plate must be washed again with an aqueous medium before examining the fluorescence of the plate. The invention also includes anti-idiotypic antibodies raised against the L6 antibody; antibodies with similar properties to the corresponding tumor antigens can be prepared (Nepom et al., (1984) Proc. Natl. Acad. Sci. 81: 2864).

To determine the presence of binding between the antibody and the cell sample when a fluorescent label is used, the plate can be examined for fluorescence, usually using a fluorescence microscope. When a label other than fluorescent is used, the formation of a precipitate, color, or the like can be examined from the plate or sample.

The above description is directed primarily to the use of the antibodies of the invention in immunofluorescence methods. However, the antibodies of the invention can be used in most assays involving antigen-antibody reactions. Assays can be homogeneous or heterogeneous. In a homogeneous analysis, the sample may be a biological fluid, such as serum, urine, and the like, or the sample may be digested and clarified by removing degradation products. In general, the immunological reaction comprises a specific antibody, a labeled analyte, and a sample of interest. The signal from the label is generated, directly or indirectly, by binding the antibody to the labeled analyte. Both the immunological reaction and the detection of its amount are performed in a homogeneous solution. Immunocernical labels that can be used include free radicals, fluorescent dyes, ent-5 enzymes, bacteriophages, coenzymes, etc.

In heterogeneous analysis, reagents are generally a sample, a specific antibody, and means for producing a detectable signal. The sample is usually placed on a support, such as a plate or slide, and contacted with the antibody in the liquid phase. The carrier is then separated from the liquid phase and either the carrier phase or the liquid phase is examined to detect the signal using means which produce such a signal. Signal 15 is proportional to the presence of analyte in the sample.

Radiolabels, fluorescent agents, enzymes, etc. are used as the means by which a detectable signal can be obtained. Examples of heterogeneous immunoassays include radioimmunoassay, immunofluorescent method, enzyme-linked immunoassay, and the like.

The above immunoassay methods are described in more detail in "Enzyme - Immunoassay", Edward T. Maggio, CRC Press, Inc., Boca Raton, Florida, 1980. See also, for example, U.S. Patents 3,690,834, 3,791,932, 3 817,837, 3,850,578, 3,853,987, 3,867,517, 3,901,654, 3,935,074, 3,984,533, 3,996,345 and 4,098,876, the list of which is not intended to be exhaustive.

The antibodies of the invention can also be used to detect metastatic occurrences in human patients with NSCLC in a manner similar to that described for malignant melanoma in J. Nucl. Med. (1983) 24: 123-129 and J. Clin. Invest. (1983) 72: 2101-2114. The antibody or fragments thereof are radiolabeled and administered intravenously to a patient, which is continuously imaged using, for example, a gamma camera or the like. Studies performed by transplanting a foreign human lung cancer cell into a thymus-deprived, "naked" mouse have shown that .3, I-labeled Fab fragments prepared from L6 are selectively localized to the transplanted tumor. This shows, based on previous studies on melanoma (J. Nucl. Med. (1983) 24: 123-129; J. Clin. Invest. (1983) 72: 2101-2114, that L6 and L6-derived tumor-selective localization of antibody fragments is likely to follow injection into human patients.

10

The invention also includes a set of diagnostic assay devices for performing the methods described above. In one embodiment, the diagnostic assay kit of parts comprising (a) a monoclonal antibody, which is further described in Example 15 above, and (b) a conjugate of a specific binding partner for the above monoclonal antibody and a label capable of producing a detectable signal. The reagents may also contain additives such as buffers and protein stabilizers, e.g. polysaccharides and the like. The diagnostic assay kit may further comprise, if necessary, other components of the signal generating system in which the label is part, agents for reducing background interference in the assay, control reagents, test equipment, and the like. In another embodiment, the diagnostic assay kit comprises ·; a conjugate of a monoclonal antibody of the invention and a label capable of producing a detectable signal. The above additives may also be present.

The monoclonal antibodies of the invention also recognize antigenic determinant sites associated with other cancers, such as breast cancers. As a result, the present antibodies can be used in diagnostic products directed against such cancerous tumors.

The essential features of the invention are set out in the appended claims.

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eXAMPLES

The invention is further illustrated by the following illustrative examples. First, a few methods used are described.

5 Immunohistological method For immunohistological experiments on frozen bodies, the Sternberger unlabeled antibody method described in Immunochemistry was used.

John Wiley & Sons, New York, 1979, pp. 104-169, modified by 10 Garrigues et al. as in Int. J. Cancer (1982) 29: 511-515. Target tissues for these experiments were surgically obtained and frozen within 4 h of removal in iso-pentane precooled with liquid nitrogen. The tissues were then stored in liquid nitrogen or at 15-70 ° C until use. Rabbit anti-mouse IgG (Sternberger-Meyer Immunochemicals, Inc., Jarettsville, MD) was used at a dilution of 1/50. A mouse peroxidase-anti-peroxidase complex (PAP, Sternberger-Meyer Immunochemicals, Inc.) containing 2 mg / ml of specially purified 20 PAP was used at a dilution of 1/80. Frozen pieces were prepared, dried, treated with acetone, and dried (Garriques et al., 1982). Sections used for histological evaluation were stained with hematoxylin. To reduce the nonspecific background, the pieces were preincubated with 25 ordinary human serum diluted 1/5 (Garroques et al., 1982). Mouse antibodies, goat anti-mouse IgG, and mouse PAP were diluted with a solution of 10% normal human serum and 3% rabbit serum.

The staining method involved treatment of a set of pieces with either a specific or control antibody for 2.5 h, incubation with rabbit anti-mouse IgG at a dilution of 1/50 for 30 min, and exposure to the mouse PAP complex diluted 1/80 for 30 min. After each antibody treatment, the ob-35 plates were washed twice with phosphate buffered saline (PBS). The immunohistochemical reaction was developed with freshly prepared 0.05% 3,3'-diaminobenzidine tetrahydrochloride (Sigma, St. Louis, MO) and 94291 16

With 0.01% hydrogen peroxide in 0.05 M Tris buffer, pH

7.6 for 8 min. Additional exposure to a 1% solution of OSO 4 in distilled water for 20 min deepened the color. The pieces were rinsed with water, dehydrated with alcohol, clarified with xylene and fixed on slides.

Each slide was coded and the coded samples were examined using independent researchers. Typical object plates were photographed using separation interference contrast-10 optics (Zeiss-Nomarski). The degree of antibody staining was estimated to be 0 (no reactivity), + (few positive cells), ++ at least one third of the cells positive), +++ (most cells positive), ++++ (almost all cells strongly positive). Because the differences in staining between + and 0 staining were less pronounced than between ++ and + staining, it was decided to calculate a staining degree of ++ or greater as positive. Tumor samples were examined for both neoplastic and basal tissue cells; the observed staining was compared to the staining of tumor cells because basal tissue-20 cells did not stain at all or stained less well than tumor cells.

Determining the location of antigens

The intracellular location of antigens was determined by measuring the binding of 25 antibodies to cells before or after permeabilization with a nonionic detergent. Antibodies that bound to the surface of intact cultured cells were identified by either direct binding assay using an I25I-labeled antibody (Brown et al., Proc.

30 Natl. Acad. Sci. U.S. (1981) 28: 539-543) or by indirect fluorescence using a (FACS) II cell sorter. Antibodies that bound to intracellular sites were determined by direct binding of 125 I-labeled antibody to cells, followed by attachment with paraformaldehyde 35 and further permeabilization with the non-ionic detergent NP-40.

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Binding Assays a) For binding assays performed using radiolabeled antibodies (Brown et al., Supra), cultured cells (106) were incubated at 4 ° C for 30 min at 106 cpm with 125 I-labeled antibody 100 heat-activated (30 min, at 56 ° C) fetal calf serum in culture medium at μΐ. After the addition of 5 ml of PBS, the cells were pelleted by centrifugation at 250 x g for 10 min. The supernatant was aspirated off and 125 I was measured from the pellet. To measure non-specific binding, co-incubations were performed using 10 μg of unlabeled antibody as a competitor (Brown et al., Supra). In some cases, binding assays were performed in an analogous manner using a cell layer attached to plastic culture dishes.

B) For binding assays performed on a FACS II cell sorter, cells were removed from the substrate using PBS containing 5 mM ethylenediaminetetraacetic acid (EDTA). Samples containing 1 x 10 5 cells were first incubated with monoclonal antibody at a concentration of 2 μg / ml and then with fluorescein-bound goat anti-mouse antibody at a dilution of 1: 200. The cells were then washed and resuspended in medium. Immediately prior to FACS analysis, propidium iodide was added to a final concentration of 1 μg / ml to stain dead cells. During FACS analysis, cells emitting red fluorescence were electronically excluded so that only living cells were detected. The average fluorescence intensity of fluorescein was then determined for each antibody. Negative controls included samples from which the monoclonal antibody had been omitted; positive controls comprised monoclonal antibodies against antigens of histocompatible HLA type 1. Staining was considered positive if the mean channel fluorescein was at least 3 times the background size.

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Determination of protein antigens

To identify protein antigens, the surface of lung cancer tumor cells was radioiodinated or metabolically labeled with 33S-methionine. Antigens were isolated from cell lysates by incubation with a monoclonal antibody by the addition of goat anti-mouse IgG and adsorption to S. au. Immunoprecipitates were washed and analyzed by sodium dodecyl sulfate polyacrylic gel electrophoresis (SDS-PAGE) (10-20% acrylamide) as described (Brown et al., Supra).

Determination of antibody reactivity to glycolipids_

The reactivity of the antibodies to glycolipid antigens was tested by incubating the antibodies with purified glycolipids adsorbed on the wells of a microtest plate (with cholesterol and lecithin) and a thin layer with chromatography plates to which the glycolipids had been fractionated. Bound antibody was determined by incubating mouse immunoglobulin with antiserum and radioiodinated protein A.

Isotyping a) Ouchterlony immunodiffusion A 25 aliquot of the supernatant of specific hybridoma cells was placed in the middle well of a 2% agar plate. Monospecific rabbit anti-mouse Ig isotype antibodies (Melody) were placed in the outer wells and the plate was incubated for 2 h at room temperature overnight at 4 ° C.

, b) Flexible 96-well polyvinyl chloride plates (Costar) were coated with 0.1 mg / ml goat anti-mouse Ig antibody for 2 h at 37 ° C and further coated with 35% BSA solution. 2 h at 37 ° C. The hybridoma supernatant was then incubated at 37 ° C for 2 h. After washing with PBS bovine serum albumin (BSA), the plates were incubated at 37 ° C for 2 h with monospecific rabbit anti-mouse Ig-94291 19 isotype antibodies bound to peroxidase (Zymed). After washing, the plate was incubated at 1 mg / ml with orthophenylenediamine and 0.03 H 2 O 2 in 1 M citrate buffer, pH 4.5. The optical density at 630 nm was determined on a Dynatec ELISA plate reader.

Staphylococcal Protein A Binding Assay Wells of a microtiter plate were incubated with 5% MCS in PBS and 0.02% NaN3 and the supernatants were aspirated. 25 μl of tumor cell suspension (2 x 10 7 cells / ml) was added to each well and incubated with 25 μl of specific antibody for 1 h at room temperature. The plates were centrifuged at 1200 rpm for 7 min, washed twice with 50% NCS / PBS / NaN3, and 25 μl of 125 I-15 staphylococcal protein A (approximately 50,000 cpm / 25 μΐ) was added. The plates were incubated for 1 h at 25 ° C, washed twice with 5% NCS / PBS / NaN 3 and dried. The bottoms of the wells were cut off and measured with a gamma counter.

20 Example

Preparation of Monoclonal Antibodies Monoclonal antibodies were produced by immunizing 3-month-old BALB / c mice with a single human tissue from four different sources: (1) pleural effusions from patients with metastatic non-small cell lung cancer, (2) cultured non-small cell lung cancer tumor and (3) lung tissue from 3-4 month old human fetuses. Immunization was performed by injecting mice intraperitoneally 3-4 times with approximately 107 cells. Three days after the last immunization, the spleens were removed, suspended in medium, and fused with NS1 mouse myeloma cells (Köhler and Millstein, supra). The mixtures were inoculated to obtain low-density cultures from single-fused cells (clones): the technique used for hybridization has been previously described by Yeh, et al., Int. J. Cancer (1979) 29: 269-275.

94291 20

Hybrid cell supernatants were screened using both ELISA and autoradiographic indirect 125 I-labeled protein A assay (Brown et al., J. Immunol. Meth. (1979) 31: 201-209) against tumor extracts used for immunization, which included e.g. . cell membranes. These extracts were prepared using the method modified by Colcher et al., Cancer Res., (1981) 42: 1451-1459; Yeh et al., Supra. To this end, the mixtures were washed with PBS and suspended, which was done on intact tumors by sifting them through a stainless steel sieve. Then, 1 mM NaHCO 3 containing 1 mM phenylmethylsulfonyl fluoride (Calbiochem-Behring Corp., San Diego, CA) was added, and then the material was homogenized on ice by pushing the B-pest of a 50 Dounce homogenizer. After centrifugation at 27,000 x g for 15 min, the supernatant was removed and the pellet was resuspended in PBS, kept in an ultrasonic bath for 1 min and stored at -70 ° C.

Hybridomas producing antibodies that bind to cell membrane extracts were cloned, grown in vitro, and further tested for antibody specificity. This testing was performed using the immunohistological method described above, which tested the ability of the antibodies to bind to frozen lung cancer tumor fragments, other tumors, and normal human tissues. Those hybrid domains that produced antibodies with apparent specificity against human lung cancer were recloned, grown, and injected into intact, selected 3-month-old BALB / c mice in which they grew as aquatic tumors. Antibodies secreted into the stomach were purified by protein A-Sepharose (Ey et al., Immunochemistry (1978) 15: 429) or by gel filtration on Sephacryl S-300. Purified antibodies were further used for characterization, which included novel immunohietology specificity assays, binding assays to intact cells to determine which antibodies

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94291 21 bind to the cell surface, and the radioimmunoassay assays described above.

Monoclonal antibody L6 was produced from hyb-5 ridoma, respectively, as described above. This antibody had the properties described above in this specification. The cell line, designated L6, was deposited with the ATCC on December 6, 1984 and assigned accession number HB 8677.

10 Example

Reactivity of Certain Glycolipids with L6 Antibody L6 antibody reactivity assays were performed according to standard methods as described by Kannagi et al., (1983) Cancer Research 43: 4997-5005 and Nudelman et al., (1982) J .

15 Biol. Chem. 257: 12752-12756. Certain glycolipids were analyzed for reactivity with the L6 antibody.

Glycolipids separated by TLC were immunostained with antibody and glycolipids were subjected to solid phase radioimmunoassay attached to the surface of plastic wells. The results are summarized in Table i.

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Table I

Structure Reactivity with L6

Asialo GalNAc-GM,

GalNAcfil-> 4GalSl-> 3GalNAcSl-> 4GalSl-> 4GlcSl-> lCer +++ 5 -

cross reactions

Gg3 (asialo GM2)

GalNAcSl-> 4GalSl-> 4GlcSl-> lCer ++

10 Gg4 (asialo GMJ

GalSl-> 3GalNAcSl-> 4GalSl-> 4GlcSl-> lCer + x2 glycolipid

GalNAcSl-> 3GalSl-> 4GlcNAcSl-> 3GalSl-> 4GlcSl-> lCer ± 15

Gb3 (CTH; pk antigen)

Galal-> 4GalSl-> 4GlcSl-> lCERs

Globoside (P-antigen) 20 GalNAcSl -> 3Galoil -> 4GalSl -> 4GlcSl -> lCer

Paraglobosidi

GalBl-> 4G1cNAcSl-> 3GalS1-> 4GlcSl-> lCer 25 5 other glycolipid structures L6 antibodies reacted specifically with asialo GalNAc-GM2 and asialo GalNAc-GM, the strongest reaction was observed with asialo GalNAc-GM1. Poor reactivity was observed with gangliotetra-30-osylceramide (asialo GM1 and Xj-glycolipid (structure shown in Table I). with the ganglio and lactose series structures were all -35 negative, which is due to the fact that the L6 antibody recognizes the following sequence:

GalNAcSl-> 4GalSl-> 3GalNAcSl-> 4GalSl-> R, where R is an unspecified carbohydrate.

Il 94291 23

Example

Specificity by immunohistology testing against different tumors_

Tissue Antibody L6 5 Lung cancer Adeno 18/19 scaly 8/10 small cell 2/6 large cell 2/2

Breast cancer 13/16 10 Colon cancer 9/9

Melanoma 1/4 "Other tumors" (sarcoma, etc.) 1/9

The invention has been described in detail, in particular with reference to the above embodiments. It is to be understood, however, that deviations and modifications may be made within the spirit and scope of the invention.

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Claims (7)

1. Monoclonal antibody, characterized in that its binding site for the antigen binds to (a) a carbohydrate antigen similar to ganglio-N-triocylceramide, which is labeled asialo-GM surface determinant of human non-small cell lung carcinoma; and (b) a protein antigen whose molecular weight is about 20,000 daltons and which is associated with human non-cellular lung carcinoma. 2. Monoclonal antibody to antigens of human ic-non-cellular lung carcinoma, characterized in that it is produced by hybridomas having deposit number ATCC 15 HB 8677. Monoclonal antibody according to claim 1 or 2, characterized in that the monoclonal antibody comprises a Fab, F (ab ') 2 or Fv fragment thereof. 20 Monoclonal antibody according to claim 1 or 2, characterized in that the monoclonal antibody is conjugated with a stamp capable of producing a detectable signal. Monoclonal antibody according to claim 4, characterized in that the stamp is a fluorescent substance, radio stamp, chromophore or enzyme. The monoclonal antibody according to claim 3, characterized in that the fragments of the monoclonal antibody are conjugated with a stamp capable of producing a detectable signal. 7. Monoclonal antibody according to claim 6, characterized in that the stamp is a fluorinating substance, radio stamp, chromophore or enzyme.