AU601680B2 - Monoclonal antibodies for human non-small cell lung carcinomas - Google Patents

Description

AUSTRALIA

601680 Patents Act COMPLETE SPECIFICATION

(ORIGINAL)

Class Int. Class Application Number: 515. 1 1 %S Lodged; Complete Specification Lodged; 40 *0 P 'riority 0 0 ea« 0 Related Art: Accepted: Published: *9 9 6# 9*9* 4 40 Name(s) of Applicant(s): 0r* Addrcss(es) of Applicant(s), 9t 1 Aetual Inventor(s): APPLICANT'S REP,: 5624-020-007 INGEGERD IIELLSTROM, KARL ERIK Hi,.LLSTROM, JOSEPH P. BROWN and DIANE HORN 132/em PETER LINSLEY, 3925 North East Surber Drive, Seattle, Washington, United States of America; 3925 North East Surber Drive, Seattle, Washington, United States of America; 243o Ninth West Street, Seattle, Washington, United States of America; 409 Newton Street, Seattle, Washington, United States of America; and 202 West Olympic Place #404, Seattle, Washington, United States of America.

as above Address for Service Is: PHILLIPS, ORMONDE AND FITZPATRICK Patent and Trade Mark Attorneys 367 Collins Street Melbourne, Australia, 3000 Complete Specification for the invention entitled: MONOCLONAL ANTIBODIES FOR HUMAN NON-SMALL CELL LUNG CARCINOMAS The following statement Is a full description of this invention, including the best method of performing it known to applieant(s): mw011A41 i I i -le- MONOCLONAL ANTIBODIES FOR HUMAN NON-SMALL CELL LUNG CARCINOMAS BACKGROUND OF THE INVENTION 151. Field of the Invention Human lung carcinomas are responsible for most deaths from cancer among men and are in the process of overtaking breast carcinomas as the most frequent cause of cancer death among women (Cancer Facts and Figures, 201983). This disease can be divided into 4 major histological types, epidermoid adenocarcinoma large-cell undifferentiated and small-cell Most cases of lung carcinomas are incurable by Zhemotherapy and radiation therapy. Small 25 cell lung carcinomas may respond to chemotherapy and radiatio'i therapy by a reduction in size, but not a total cure. Complete surgical removal of the tumor appears to be the only effective therapy. Unfortunately, however, fewer than 30% of lung cancer patients have tumors which 30 can be totally resected at diagnosis and of these, fewer than one-third survive 5 years after apparent complete surgical removal of all tumor. There is a great need, therefore, for methods that would make possible an 9558K 24730 -"q -2earlier diagnosis of lung cancer, a better definition of the degree of cancer spread, and a more effective therapy.

Monoclonal antibodies may be used for all these purposes.. A prerequisite, however, is to find antibodies to antigens that are more strongly expressed in lung cancer than in normal adult tissues. In view of the known heterogeneity of tumor cell populations, the presence of several determinants on the same antigen molecule, the anticipated differences between antigens respect to their suitability as diagnostic markers as compared to therapeutic targets, and the different biological characteristics of different antibodies to the same antigen, a number of different antibodies may be needed.

152. Description of tihe Prior Art Human monoclonal antibodies to lung cancer antigens are described by Sikora et al., Br. 3. Cancer (1981) 43:696-700. Monoclonal antibodies that demonstrate specificity for several types of human lung cancer are ir 20disclosed by Cuttitta et al., Proc. Natl. Acad. Sci.

U.S.A. (1981) 78:4591-4595. Antigens associated with a human lung adqnocarcinoma defined by monoclonal rntibodies are described by Varki et al., Cancer Research (1984) 44:681-687.

25 Mouse monoclonal antibodies to glycolipid LeX antigens are described by Hakomori et al. in Biochemical and Biophysical Research Communications (1981) 100: 1578-1586; Brockhaus et al., Arch. Biochem. Biophys.

(1982) 217:647; Fukushi et al., J. Biol. Chem. (1984) 30259:506; Fukushi et al. 3. Exp. Med. (1984) 159:506; Chia et al., Cancer Res. (1985) 45:435. Antibodies to Ley antigens have been described by Abe et al., 3. Biol. Chem (1983) 258:8934; Lloyd et al., Immunogenetics (1983) 17:537; Brown et al., Biosci. Rep. (1983) 3:163.

9558K 24730 i-i- ~~i-aYuf~arno~aJlafirsMc~ F -3- Continuous cultures of fused cells secreting antibody of predefined specificity are described by Kdhler et al., Nature (1975) 265:495-497.

SUMMARY OF THE INVENTION The present invention is concerned with novel monoclonal antibodies which define determinant sites on carbohydrate (glycolipid) antigens associated with human non-small cell lung carcinoma (NSCLC) cells. The term cells" includes epidermoid carcinoma cells, adenocarcinoma cells, and large cell undifferentiated carcinoma cells. The determinant site may also be found on antigens of some other carcinomas, some carcinomas of the breast, and, thus, the antibodies of 15 the invention will also bind to these other carcinoma cells. The present monoclonal antibodies bind to a much lesser degree to normal adult cells than to tumor cells.

The term "bind to a much lesser degree" means that the binding will not be detectable by immunohistological and that there will be much weaker binding to normal cells than to tumor cells. The monoclonal antibodies are secreted by murine hybridomas.

The present invention also includes methods for determining the presence of a malignar.t condition in the by examining lung tissue and other human tissue for the presence of an antigen having the characteristics of Le or Le y antigens. The Lex antigen has been described by Hakomori et al., supra; Brockhaus et al., supra; Fukushi et al., supra; and Chia et al., supra; The antigen has been described by Abe et al., supra, Lloyd et al., supra, and Brown et al., supra.

For example, tissue from a subject can be contacted with an antibody which defines a determinant site on a cell associated carbohydrate antigen which is Le y or 9558K 24730 i i t 'C t

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t C C JU *t t 4 I CC .t -4- LeX or which has the characteristics of Le Y or Le X or a functional equivalent or fragment of such antibody.

Thus, the invention concerns certain diagnostic methods employing the monoclonal antibodies of the One such method involves the determination of the presence of NSCLC cells in a specimen suspected of containing such cells. The specimen is contacted with the monoclonal antibody, which is capable of distinguishing such cells from other cell types which may lobe present in the specimen. The contact is carried out under conditions for binding of the antibody to such cells. After contact, the presence or absence of binding of the antibody to the cells in the specimen is determined. This binding is related to the presence or i5absence of the NSCLC cells in the specimen. Generally, the specimen is contacted with a labeled specific binding partner for -the monoclonal antibody. This label is capable of producing a detectable signal. Another diagnostic method involves the localization to a tumor of 2Oantibodies or an~tibody fragments which have been properly labelled with a radioisotope) ardi are subsequently injected into.patients. This method can provide better ways to stage cancer patients with respect to extent of disease and to monitor changes in response to therapy.

25 The invention also has therapeutic applications.

The antibodies can react with either or both of the above-mentioned antigens that are expressed in high concentrations at the tumor cell surface. It can be used as a carrier of various agents which have an anti-tumor 30 effect, including, but not restricted to, chemotherapeutic drugs and radioisotopes.

DESCRIPTION OF THE SPECIFIC EMBODIMENTS The present invention concerns novel antibodies 35 which bind to an antigen on human NSCLC cells and certain 58K273 24730

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diagnostic and therapeutic methods employing the-e antibodies. The monoclonal antibodies of the invention may be produced according to the standard techniques of Kbhler and Milstein, supra. For example, human lung cells from plural effusions or cultured cells from human non-small cell lung carcinoma, or cells from a normal fetal lung, are used as the immunogen. These cells are injected into a mouse and, after a sufficient time, the mouse is sacrificed and spleen cells obtained.

spleen cell chromosomes encoding desired immunoglobulins are immortalized by fusing the spleen cells with myeloma cells or with lymphoma cells, generally in the presence cf polyethylene glycol. The H resulting cells, which include the fused hybridomas, are to grow in a-selective medium, such as HAT-medium, and the surviving cells are grown in such medjum using limiting dilution conditions. The cells are grown in a suitable container, microtiter wells, and the supernatant is screened for monoclonal antibodies the desired specificity.

SVarious techniques exist for enhancing yields of monoclonal antibodies, such as injection of the hybridoma ji cells into the peritoneal cavity of a mammalian host, Swhich accepts the cells, and harvesting the ascites Where an insufficient amount of the monoclonal antibody collects in the ascites fluid, the antibody is harvested from the blood of the host. Various conventional ways exist for isolation and purification of the monoclonal antibodies, so as to free the monoclonal from other proteins and other contaminants (see K6hler and Milstein, supra).

One monoclonal antibody in accordance with the present invention is designated L15. It defines a cell surface carbohydrate antigen which is, or which has the of, Ley antigen. We have identified 9558K 24730 this antigen as characteristic of human NSCLC cells and cells from certain other human carcinomas as listed in Hellstrom et al., (1986) Cancer Research 46, 3917-3923. The antibody is of the IgM isotype. It does not bind detectably to normal cells, such as fibroblasts, endothelial cells, or epithelial cells in the major organs. The L15 antibody is produced by the L15 murine hybridoma.

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r -6cells from certain other huma arcinomas. The antibody is of the IgM isotype. does not bind detectably to normal cells, suc s fibroblasts, endothelial cells, or s in the major organs. The L15 antibody ,s piced bythe murine hybridoma.

Another monoclonal antibody in accordance with the present invention is designated L17. It defines a cell surface carbohydrate determinant which is, or which has characteristics of, Le x antigen. We have identified this antigen also as characteristic of human NSCLC cells and cells from certain other human carcinomas. This antibody is of the IgM isotype. It S ,binds less to normal cells, such as fibroblasts, T 15endothelial cells, or-epithelial cells in the major Sorgans than to tumor cells. The L17 antibody is produced by the L17 murine hybridoma.

Antibody L15 recognizes antigen Le y which has the f structure as identified below: Gal B14GlcNAc B13Gal Bl+R 2 3 Fucal Fucal A series of glycolipids with different chain lengths can be detected by this antibody.

25 Antibody L17 reacts with a series of glycolipid antigens, all of which carry Le x at the terminus, i.e.: Gal BI-4GlcNAc+R 3 Fuc tl can be various lengths of blood group type 2 chain (Galii+4GlcANc8 l+3Gal Bl4GlcNac 813).

The antibody also reacts with trifucosyl Y structure as shown below: 335 S 955K 35 S9558K 24730 -7- Gal 1-+4GlcNAc 813Gal 81a4GlcNAc 81+3Gal B1+4GlcNAc B13Gal 1 +4Glc 3 3 3 Fucal Fucdl Fucal Also included within the scope of the invention are useful binding fragments of the above monoclonal antibodies such as Fab, F(ab') 2 Fv fragments and so forth. The antibody fragments are obtained by conventional techniques. For example, useful birding fragments may be prepared by peptidase digestion of the using papain or pepsin.

The invention also includes the diagnostic and rC therapeutic use, in detecting and treating NSCLC o t carcinoma in humans, of Le or Le x antigen or antigens having the characteristics of Le and Le S 1 5 antigens, that is, antigens which are related but not identical to Le or Lex and which are recognized by an antibody that also recognizes Le or Lex. The I antigen can be purified by conventional methods such as described by Hakomori et.al., sJpra, and by Abe et al., supra.

One method of the invention involves the Sdetermination-of the presence of a malignant condition in the lung of a subject by examining lung tissue or other tissue from the subject for the presence of a antigen, which is Le y or Lex or which has the characteristics of Ley or Lex. The term "tissue" refers to excised cells, exfoliative cells, body fluids such as blood, plasma, serum, urine, etc., and the like. The term "malignant condition" refers to the I30presence of dysplastic cells including carcinoma in situ, neoplastic, malignant, or tumor cells, or the like. For example, the specimen can be contacted or combined with a monoclonal antibody which recognizes a determinant site on Le y or Lex such as a monoclonal antibody of the 35invention, as'L15 or L17 antibody or an antibody 9558K 24730

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-8having similar characteristics such as, for example, those antibodies described by Hakomori et al., supra, Fukushi et al., supra, Chia et al., supra, Abe et al., supra, Lloyd et al., supra, and Brown, et al., supra.

contact is carried out under conditions for binding of the antibody to the malignant cells. After contact, the presence of binding of the antibody to the malignant cells in the specimen is observed. That is, the specimen is examined for immune complexes of the antibody and the site. This immune complex formation is related to the presence of malignant cells in the specimen.

A particular example, by way of illustration and not r limitation, of a method in accordance with the invention is a method for the detection of tumor cells in excised 15 tissue. The above me-thod is applied to a specimen which is a section of the tumor obtained after removal of the cumor. The tumor that is excised is treated to obtain sections, which treatment initially involves freezing the tumor or tissue, normally freezing immediately after The frozen layer of tissue is then cut into sections using, for example, a cryostat.

The section of the tumor obtained as described above is contacted with a monoclonal antibody of the invention and then with a second antibody directed against the 25 above monoclonal antibody, which second antibody is labeled with a detectable label.

The excised specimen, the section of the tumor, is contacted with the first monoclonal antibody under conditions for binding of the antibody to the Sjomalignant cells. The incubation is generally conducted in an aqueous medium such as, for example, phosphate buffered saline containing a small amount of sodium azide, in a suitable container such as, for example, a glass petri dish, for a period from about 15 to 35 minutes at a temperature of from about 20 to 30 0 C. The 9558K 24730 .1 1 -9- C CC CE C C QP amount of antibody employed is usually sufficient to provide detectable binding, to provide a detectable number of immune complexes between the antibody and the determinant or antigenic site in question.

Following the incubation, the section is washed to reduce or eliminate non-specifically bound antibody and then is examined to observe the above-mentioned complexes which result from binding of the monoclonal antibody to the cells of the specimen possessing the antigenic site.

binding is related to the presence of malignant cells in the section. Accordingly, binding is determined, for example, by contacting the specimen with a labeled specific binding partner for the monoclonal antibody.

The label is capable of producing a detectable signal and may be a radioactive label, a chromophore such as a fluorescer, an enzyme, or the like.

An example of a technique employing the above approach is immunofluorescence staining. In this technique frozen sections of the tumor are fixed on a 20 glass slide with acetone and ar-e incubated with the monoclonal antibody in, for example, a petri dish. After washing with an appropriate buffer such as, for example, phosphate-buffered saline, the section is placed on a petri dish and contacted with the labeled specific partner for the monoclonal antibody, which may be, for example, a labeled antibody specific for the monoclonal antibody employed. Since, for the most part, the monoclonal antibody will be derived from a murine source, a labeled anti-mouse immunoglobulin specific for 30 the monoclonal antibody may be employed. Such immunoglobulins may be raised according to standard techniques by injecting a suitable host with murine antibody, waiting for an appropriate time, and harvesting the anti-mouse immunoglobulins from the blood of the host.

C IC f: 9558K 24730 -irr~l"~u*-r rit rurrp;~rrr-^ r i

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S* 09 o r ,9 -w a 6* 9 9$ After a second washing of the slide with, for example, an aqueous buffer, the sections may be covered with a fluorescent antibody mounting fluid and a coverslip and then examined with a fluorescence to determine the binding of the monoclonal antibody to the section. The determination of the binding also may include an identification of the location of such binding within the specimen.

The binding of the monoclonal antibody to the 10specimen may also be determined by employing a monoclonal antibody which is covalently conjugated to a label capable of producing a detectable signal, such as a radioactive entity, a chromophore including dyes and fluorescers, or an enzyme. The number of labels employed 15per antibody is generally determined by the requirements of the diagnostic method in which the labeled antibody is employed and the availability of sites for linking the label to the antibody.

Methods for conjugating labels to antibodies and fragments are well-known in the art. Such methods may be found in U.S. Patent Nos. 4,220,450; 4,235,869; 3,235,074; and 3,996,345.

Another example of a technique in which the monoclonal antibodies of the invention can be employed is 25 the detection of molecules bearing the Le x and Ley determinants in body fluids including plasma and serum.

For this purpose, the antibody of the invention may be used alone or in conjunction with another antibody directed against a separate determinant.

Still another example of a technique in which the monoclonal antibody of the invention can be employed is immunoperoxidase labeling (Sternberger, Immunocytochemistrv, John Wiley Sons, New York, 1979, pp:104-1 6 9 as modified by Garrigues et al., Int. J.

35 Cancer (1982) 29:511-515). The tissue to be tested is 9558K 24730 ~i -11fixed with a suitable solvent, such as acetone, on a support, such as a glass slide. Next, the tissue is incubated with the monoclonal antibody and then washed free of unbound antibody. Then, the tissue is incubated with rabbit anti-mouse IgG, washed to remove unbound antibody, combined with mouse peroxidase-anti-peroxidase I complex, washed to remove unbound conjugate, and then treated with substrate for the enzyme. Following this I treatment the slide is examined for a detectable signal.

The antibodies of the invention may be used in a method of determining the presence of a malignant condition, for instance in an exfoliative cell specimen from the lung, such as sputum or in a cervical smear. By the term "exfoliative" is meant that the specimen 15comprises isolated cells or clumps of cells obtained by Sscraping or washing the surface of tissue, which cells are removed individually or in scales or laminae. The exfoliative cell specimen is to be distinguished from excised tissue such as that obtained by biopsy. Contact the specimen and the antibody is made under conditions for binding of the antibody to the antigenic site. After contact, the presence or absence of binding of the antibody to the antigenic site is determined and is related to the presence of a malignant condition in lung.

To determine the presence of a malignancy in the lung, a sputum sample would provide the exfoliative cell specimen to be used in the method. The method may find utility in the detection of a malignant condition in cell specimens from the bronchus, gastro-intestinal tract including oral pharynx, mouth, The exfoliative cell specimen is next contacted with the aforementioned antibodies under conditions for 36binding of the antibody to the specific antigenic site in 9558K 24730 -f ij-ii i

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3- 1 I -12the specimen to form antigen-antibody complexes. This antigenic site may be present on cells or cell fragments in the specimen. Generally, the specimen is placed on an i appropria.te support, such as, for example, a slide, glass, or some other suitable material. The exfoliative cell specimen is generally smeared on the sltie to provide a thin layer of the specimen on the s ice of the slide. The contact between the antibody anc e specimen is generally carried out in an aqueous 10 bufr' medium. The buffers which may be employed includ. osphate, tris, bicarbonate, etc. The pH is relaed to the nature of the specimen and the antibody, and is generally in the range of from about 5 to 8. The aqueous medium may additionally contain organic polar t 15solvents in an amountof from about 0 to 40%. The organic polar solvents are water soluble and generally have from about 1 to 10 carbon atoms and from about 1 to 4 oxygen atoms. The antibody will be present in the aqueous medium at a concentration of about 1 to 0100 vg/ml, preferably from about 10 to 20 ug/ml. The temperature during the contact of the specimen with the antibody is usually from about 4 to 400C, preferably about 10 to 30°C. The period of contact is usually from about 15 to 120 minutes, preferably from about 30 to 25 minutes.

After the period of contact between the specimen and the antibody, the support is generally treated to remove unreacted antibody. Normally, this is accomplished by washing the support with an aqueous, usually buffered, 3 dnedium. In general, the amount of wash solution should be sufficient to remove the unreacted antibody.

Next, the presence of antibody bound to the antigenic site in the specimen, which binding is related to the presence of a malignant condition at the locus, is 35 observed. That is, the specimen is examined to determine 24730 ~k7 -7 the number of antigen-antibody (immune) complexes formed. It should be noted that in some instances very small numbers of the antigenic site in question may be found inthe exfoliative cell specimen. However, in a Smalignant condition, large numbers of the antigenic site will be present and this latter condition is readily distinguishable by this method over a non-malignant condition because a large number of antigen-antibody complexes will be measurable where a malignant condition To make the determination of the-presence of binding, means for producing a detectable signal is incorporated into the assay system. For example, one may conjugate the antibody employed in the assay to a label which is capable of producing a detectable signal. The 151abel may be a radioactive entity, a chromophore including dyes and fluorescers, an enzyme, or the like.

The number of labels employed for the antibody is generally determined by the requirements of the method and the availability of sites for linking the label to antibody.

Alternatively, one may contact the washed slide with.

a labeled specific binding partner for the antibody, which may be, for example, a labeled antibody specific for the antibody employed. Where the monoclonal antibody derived from a murine source, a labeled anti-mouse immunoglobulin specific for the antibody employed in the method may be used. Such immunoglobulins may be raised according to standard techniques by injecting a suitable host with the monoclonal antibody, waiting for an time, and harvesting the anti-mouse immunoglobulins from the blood of the injected host.

When a labeled specific binding partner for the antibody is employed, the slide must be washed again with an aqueous medium prior to examining the slide for i: t 9558K 24730 c--j -14- To determine the presence of binding between the antibody and the cell specimen where a fluorescer label is used, one may examine the slide for fluorescence, usually employing a fluorescence microscope. Where a label other than a fluorescer is employed, one may examine the slide or the specimen for the formation of a precipitate, a color, or the like.

The above description is directed primarily to the use of the antibodies of the invention in techniques. However, the antibodies of the invention can be used in most assays involving antigen-antibody reactions. The assays may be homogeneous or heterogeneous. In a homogeneous assay approach, the specimen may be biological fluid such as 15 serum, urine, and the.like or the specimen may be lysed and clarified to remove debris. The immunological

I

s reaction usually involves the specific antibody, a labeled analyte, and the sample of interest. The signal arising from the label is modified, directly or upon the binding of the antibody to the labeled analyte. Both the immunological reaction and detection of the extent thereof are carried out in a homogeneous solution. Immunochemical labels which may be employed include free radicals, fluorescent dyes, bacteriophages, coenzymes, and so forth.

In a heterogeneous assay approach, the rea'gents are usually the specimen, the specific antibody, and means for producing a detectable signal. The specimen is generally placed on a support, such as a plate or a 30 slide, and contacted with the antibody in a liquid J* 1 phase. The support is then separated from the liquid phase and either the support phase or the liquid phase is examined for a detectable signal employing means for producing such signal. The signal is related to the of the analyte in the specimen. Means for 9558K 24730 j 7 t producing a detectable signal includes the use of radioactive labels, fluorescers, enzymes, and so forth.

Exemplary of heterogeneous immunoassays are the radioimmunoassay, immunofluorescence methods, enzyme-linked immunoassays, and the like.

For a more detailed discussion of the above immunoassay techniques, see "Enzyme-Immunoassay," by Edward T. Maggio, CRC Press, Inc., Boca Raton, Florida, 1980. See also, for example, U.S. Patent Nos. 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, which listing is not intended to be exhaustive.

The antibodies of the invention can also be employed to image metastatic deposits in human patients with NSCLC in a manner analogous to that described for malignant melanoma in J. Nucl. Med. (1983) 24:123-129 and in J.

Clin. Invest. (1983) 72:2101-2114. The antibody or fragments thereof are radiolabelled and administered intravenously to a patient who ubsequently is imaged using, for example, a gamma camera or the like.

While the above specific examples of methods in accordance with the present invention employ antibodies binding to specific determinant sites on the respective antigens and being of the IgM sub-classes from a murine this is not meant to be a limitation. The above antibodies and those antibodjes having functional equivalency with the above antibodies, whether from a murine source, other mammalian source including human, or other sources, or combinations thereof can be employed in 30 the method of this invention, as well as other isotypes.

By the term "functional equivalency" is meant that the antibody is capable of binding to either of the respective above-described determinant sites and capable of competing with a particular antibody of the invention 35 for such site. That is, such antibody, when combined L t 9558K 24730 -16with a specimen containing a cell or cell fragment or secreted antigen having such determinant site, will bind to such determinant site and will block an antibody of the invention from binding to such site. Furthermore, the 'Le and Le x antigens can have more than one determinant site, the method of the invention includes the use of monoclonal antibodies which define determinant sites other than determinant sites defined by the aforementioned monoclonal antibodies.

The invention also includes diagnostic kits for carrying out the methods disclosed above. In one sembodiment, the diagnostic kit comprises a monoclonal antibody more specifically defined above and a conjugate of a specific binding partner for the above antibody a-nd a label capable of producing a detectable signal. The reagents may also include ancillary agents such as buffering agents and protein stabilizing agents, polysaccharides and the like.

The diagnostic kit may further include, where necessary, members of the signal-producing system of which system the label is a member, agents for reducing background interference in a test, control reagents, apparatus for conducting a test, and the like. In another embodiment, the diagnostic kit comprises a 25 conjugate of a monoclonal antibody of the invention and a label capable of producing a detectable signal.

Ancillary agents as mentioned above may also be present.

The antibodies of the invention may be used therapeutically. Antibodies with the proper biological can be useful directly as therapeutic agents t Sears, et al, Contrl. Oncol. Karger, Basel, (1984) 19:180-192). Furthermore, antibodies can be bound to a toxin to form an immunotoxin or to a radioactive material or drug to form a radiopharmaceutical or pharmaceutical.

35 Methods for producing immunotoxins and 9558K 24730 |j L_ j -17radiopharmaceuticals of antibodies are well-known (see, for example, Cancer Treatment Reports (1984) 68:317-328).

Another therapeutic use of the monoclonal antibody of the present invention is the immunization of a patient an anti-idiotypic antibody raised by using one of the present monoclonal antibodies as an immunogen. Such immunization can induce an active anti-tumor activity (see, for example, Nepom et al.; Proc. Natl. Acad. Sci.

U.S.A. (1984) 81:2864-2867. In a similar approach, the can be immunized with the respective antigen in purified form or a modified form of the respective antigens.

An attractive aspect of the present invention is S• that the present antibodies can be combined with other -15. antibodies to NSCLC such as those disclosed in U.S.

Patent Application Serial Nos. 667,521 and 684,759, filed November 2, 1984 and December 21, 1984, respectively.

e The combination is effective in detecting at least the four types of lung carcinomas mentioned above, namely, cell undifferentiated lung carcinoma, small cell lung carcinoma, adenocarcinoma, and epidermoid carcinoma.

The monoclonal antibodies of the invention also define determinant sites on antigens associated with Sother carcinomas such as breast carcinomas.

25 Consequently, the present antibodies can find use in diagnostic and therapeutic products directed to such carcinomas.

EXAMPLES

The invention is further demonstrated by the *st following illustrative Examples. A number of procedures employed will be described first.

Immunohistoloaical Technioue For immunohistological studies on frozen sections, 35 the unlabelled antibody technique of Sternberger in 9558K 24730 irg I" -18-

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cc c t 0n t ~s .trt

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Immunochemistry, John Wiley Sons, New York, 1979, pp:104-169, as modified by Garrigues et al. in Int. J.

Cancer (1982) 29:511-515, was used. The target tissues for these tests were obtained at surgery and frozen 4 hr of removal in isopentane which had been precooled in liquid nitrogen. Tissues were then stored in liquid nitrogen or at -700C until use. Rabbit anti-mouse IgG (Sternberger-Meyer Immunochemicals, Inc., Jarettsville, MD) was used at a dilution of 1/50. Mouse complex (PAP, Sternberger-Meyer Immunochemicals, Inc.) containing 2 mg/ml of specifically purified PAP was used at a dilution of 1/80. Frozen sections were prepared, dried, treated with acetone and dried (Garrigues et al., 1982). Sections to be used for 15 histologic evaluation were stained with hematoxylin. To decrease nonspecific background, sections were preincubated with normal human serum diluted (Garrigues et al., 1982). Mouse antibodies, goat anti-mouse IgG, and mouse PAP were diluted in a solution 20 of 10% normal human serum and 3% rabbit serum.

The staining procedure consisted of treating serial sections with.either specific or control antibody for 2.5 hr, incubating for 30 min with rabbit anti-mouse IgG diluted 1/50, and exposing for 30 min to mouse PAP oiluted 1/80. After each treatment with antibody, the slides were washed twice in phosphate buffered saline (PBS). The immunohistocnemical reaction was developed with freshly prepared 0.05% 3,3'-diaminobenzidine tetrahydrochloride (Sigma, St.

30Louis, MO) and 0.01% hydrogen peroxide in 0.05 M Tris buffer, pH 7.6 for 8 min. Further exposure to a 1% Os 4 solution in distilled water for 20 min intensified the stain. The sections were rinsed with water, dehydrated in alcohol, cleared in xylene, and mounted on ~i 1~3tj i 9558K 24730 C. e *e P oe Pt P i PP*

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ft 9PP

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SeP b T* e P P 4 P* 4 J

PP

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ii PP* -19- The slides were each read under code and coded samples were checked by an independent investigator.

Typical slides were photographed by using differential interference contrast optics (Zeiss-Nomarski). The of antibody staining was evaluated as 0 (no reactivity), (few positive cells), (at least one third of the cells positive), (most cells positive), (close to all cells strongly positive). Since differences between and 0 staining were less clear cut 10 than between and staining, it was decided to count as "positive" a staining graded as or greater. Both neoplastic and stroma cells were observed in tumor samples; the staining recorded referred to that of the tumor cells, since the stroma cells were not stained at 15all, or were stained more weakly than the tumor cells.

Determination of Antigen Location The subcellular localization of antigens was determined by measuring antibody binding to cells before or after permeabilization with non-ionic detergent.

20 Antibodies binding to the.-cell surface of intact cultured cells were identified by either direct binding assays with 125 1I-labelled antibody (Brown et al., Proc. Natl.

Acai. Sci. U.S.A. (1981) 78:539-543) or by indirect fluorescence using the (FACS) II cell sorter. Antibodies 25 binding to intracellular locations were determined by direct binding of 125 I-labelled antibody to cells following fixation with paraformaldehyde and subsequent permeabilization with the non-ionic detergent Binding Assays 3 0 a) For binding assays performed by using radiolabelled antibodies (Brown et al., supra), cultured cells (10 6 were incubated at 4°C for 30 min with O10 cpm of 125 1-labelled antibody in 100 pl of heat-activated min at 56°C) fetal calf serum in culture medium.

35 After the addition of 5 ml of PBS, the cells were 9558K 24730 r pelleted by centrifugation for 10 min at 250 X g. The supernatant was aspirated, and the pellet was counted for 125I. To measure nonspecific binding, parallel incubations were performed with 10 pg of unlabelled antibody as a competitor (Brown, et al., supra). In some instances binding assays were carried out in an analogous fashion on cells monolayers attached to plastic culture dishes.

b) For binding assays performed on the FACS II cell cells were removed from their substrata using PBS containing 5 mM ethylenediamine tetraacetic acid (EDTA).

Samples containing 1 x'10 cells were incubated first with monoclonal antibody at a concentration of 2 pg/ml followed by fluorescein-conjugated goat anti-mouse at a 1:200 dilution. Cells were then washed and resuspended in culture medium. Immediately prior to FACS analysis, propidium iodide was added to a final concentration of 1 pg/ml to stain non-viable cells.

During FACS analysis, cells emitting red fluorescence electronically gated out so that only viable cells were examined. The mean intensity of fluorescein S- fluorescence was then determined for each antibody.

Negative controls consisted of samples in which monoclonal antibody was omitted; positive controls of monoclonal antibodies to HLA type _1 histocompatibility antigens. Staining was regarded as positive if the mean channel fluorescein was at least 3 times background.

Determination of Reactivity of Antibodies to Glycolipids Antibodies were tested for reactivity to glycolipid 7rj antigens by incubation with purified glycolipids adsorbed to microtest wells (along with cholesterol and lecithin) and with thin layer chromatography plates on which glycolipids had been fractionated. Bound antibody was 9558K 24730 -9 3, -21detected by incubation with antiserum to mouse immunoglobulin and radioiodinated protein A.

Isotype Determination a) Ouchterlony immunodiffusion An aliquot of supernatant of particular hybridoma cells was placed into the center well of a 2% agar plate. Monospecific rabbit anti-mouse Ig isotype antibodies (Meloy) were placed in the outer wells and the plate was incubated for 2 hr at room temperature and at b) Flexible polyvinylchloride 96 well plates (Costar) were coated with 0.1 mg/ml goat anti-mouse Ig antibodies for 2 hr at 37 0 C and countercoated with a 3% BSA solution for 2 hr at 370C. The hybridoma supernatant then incubated at_37 0 C for 2 h. After washing with PBS bovine serum albumin (BSA) plates were incubated at 37 0 C for 2 hr with monospecific rabbit anti-mouse Ig isotype antibodies coupled to peroxidase (Zymed). After washing, plates were incubated with 1 mg/ml and 0.03%'H202 in 0.1 M citrate buffer pH 4.5. Optical density at 630 nm was determined on a Dynatec ELISA plate reader.

Staphylococcal Protein A Binding Assay Microtiter wells were incubated with 5% FCS in PBS 25 plus 0.02% NaN 3 and the supernatant was aspirated.

Twenty-five pl of a suspension of tumor cells (2x10 cells/ml) were added to each well and incubated with pl of a particular antibody for 1 hr at room temperature. The plates were centrifuged at 1200 rpm for 307 min, washed twice with 50% NCS/PBS/NaN 3 and 25 ul

I

1 2 5 1-staphylococcal protein A (about 50,000 cpm/25 1) were added. The plates were incubated for 1 hr at 25 0

C,

washed twice with 5% NCS/PBS/NaN 3 and dried. The bottom of the wells were cut off and counted in a gamma 3 S9558K 24730 -22-

P

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I;

i

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EXAMPLE

Preparation of Monoclonal Antibodies Monoclonal antibodies were produced by immunizing 3-month-old BALB/c mice with cells from pleural effusions from patients with metastatic non-small cell lung carcinoma. The immunizations were performed by injecting the mice intraperitoneally 3-4 times with approximately 7 cells. Three days after the last immunization, the spleens were removed, suspended in culture medium and 10fused with NS1 mouse myeloma cells (KBhler and Milstein, supra). The mixtures were seeded to form low density cultures originating from single fused cells (clones); the techniques used for the hybridization have been previously described by Yeh, et al., Int. J. Cancer 15(1979) 29:269-275.

Supernatants from hybrid cells were screened by using both an ELISA assay and an autoradiographic indirect 12 5 I-labelled protein A assay (Brown et al., J. Immunol. Meth. (1979) 31:201:209 against extracts from tumors used for immunization which contained, i.a., cell membranes. These extracts were prepared using a procedure modi-fied from Colcher et al., Cancer Res., (1981) 42:1451-1459; Yeh et al., supra. For this, tissues were washed with PBS and suspended, which for tumors was'done by pressing through a stainless steel screen. After this 1 mM NaHCO 3 containing 1 mM phenylmethylsulfonylfluoride (Calbiochem-Behring Corp., San Diego, CA) was added, and the material was then homogenized on ice, with 50 strokes of the B pestle of a homogenizer. After centrifugation for 15 min at 27,000 x g, the supernatant was removed, and the pellet was resuspended in PBS, sonicated for 1 min, and stored at -70 0

C.

Hybridomas which produced antibodies binding to the membrane extracts were cloned, expanded in vitro, t r r cP i' 9558K 24730

II~

-23- Iand further tested for antibody specificity. This testing was carried out by using the Immunohistological Technique described above, in which the ability of the antibodies to bind to frozen sections of lung carcinomas, tumors and normal human tissues were tested. Those hybridomas which produced antibody of apparent specificity for human lung cancer were recloned, expanded and injected into pristane-primed 3-month old BALB/c mice, where they grew as ascites tumors.

Antibodies secreted into the ascites were purified on protein A Sepharose (Ey et al., Immunochemistry (1978) 15:429) or by gel filtration in Sephacryl S-300.

Purified antibodies were used for further characterization which included additional specificity by immunohistology, binding assays on intact cells to determine which antibodies bound to the cell surface, and the radioimmunoprecipitation tests as described above.

Monoclonal antibodies L15 and L17 were produced from the corresponding hybridoma as described above. These antibodies exhibited the properties indicated above in this specification.

Antibodies L15 and L17 were subjected to the immunohistological technique described above.

Antibody L15 bound strongly to NSCLC cells and did 2 5 not bind to the following cells: colon carcinoma, breast carcinoma, normal heart, leukocytes, brain, colon, kidney, spleen, skin, and liver.

Antibody L17 bound strongly to NSCLC cells, colon carcinoma cells, and breast carcinoma cells. L17 30 exhibited weak binding with leukocytes and no binding with the following cells: normal heart, brain, colon, kidney, spleen, skin, liver.

S|The cell lines, designated L15 and L17, were both deposited at the A.T.C.C. on March 1, 1985, and received i 35 accession numbers HB8738 and HB8739, respectively.

9558K 24730 VZ* tV-'V V V *i L A -24- The invention has been described in detail with particular reference to the above embodiments. It will be understood, however, that variations and modifications can be effected within the spirit and scope of the ct t 9558K 24730

Claims (28)

1. A monoclonal antibody designated L15 of the IgM isotype produced by a hybridoma cell line having the characteristics of HB 8738 as deposited with the ATCC as hereinbefore described, the antigen combining site of which defines Le y a surface carbohydrate antigen characteristic of human non-small cell lung carcinoma.

2. An Fab, F(ab') 2 or Fv fragment of the monoclonal antibody of Claim 1.

3. A monoclonal antibody or fragment of Claim 1 or 2 conjugated to a label capable of producing a detectable signal.

4. A monoclonal anti ;:d3y or fragement of Claim 3 in which the label comprises a fluorescer, a radiolabel, a chromophore or an enzyme.

5. A m-noclonal antibody designated L17 of the IgM isotype produced by a hybridoma cell line having the characteristics of HB 8739 as deposited with the ATCC as hereinbefore described, the antigen combining site of which defines Le x a surface carbohydrate antigen characteristic of human non-smvall cell lung carcinoma.

6. An Fab, F(ab') 2 or Fv fragment of the monoclonal antibody of Claim

7. A monoclonal antibody or fragment of Claim 5 or 6 conjugated to a label capable of producing a detectable signal.

8. A monoclonal antibody or fragment of Claim 7 in which the label comprises a fluorescer, a radiolabel, a chromophore or an enzyme.

9. A cell line which produces monoclonal antibody LI5 as hereinbefore described.

A cell line of claim 9 deposited with the ATCC and assigned accession number HB 8738.

11. A cell line which produces monoclonal antibody L17 as hereinbefore described.

12. A cell line of claim 11 deposited with the ATCC and assigned accession number HB 8739.

13, A method for determining the presence of a malignant ,condition in the lung of a subject, which comprises I -26- contacting a specimen of tissue obtained from said subject with a monoclonal antibody as defined by any one of Claims 1-8 which defines a determinant site on an antigen which is Le y or Lex antigen or an antigen having the characteristics of Le y or Le x antigen under conditions for binding of said antibody to said specimen and observing the presence of binding of said antibody to said specimen, said binding being related to the presence of a malignant condition in the lung. to

14. A method of Claim 13 wherein said tissue is selected from the group consisting of excised tissue, exfoliative r* tissue, and body fluids.

A method of Claim 13 wherein the extent of binding is determined by contacting said specimen with a conjugate of a label and specific binding partner for said monoclonal I antibody, said label being capable of producing a detectable signal.

16. A method of Claim 13 wherein the monoclonal antibody is conjugated to a label capable of producing a detectable ato signal.

17. A method of Claim 15 wherein the labeled specific binding partner for said monoclonal antibody is an antibody specific for said monoclonal antibody.

18. The method of Claim 17 wherein the label is a chromophore.

19. A diagnostic kit, comprising a monoclonal antibody as defined in any one of Claims 1-8 useful for determining the presence of a malignant condition in the lung, said antibody defining a determinant site on a cell surface carbohydrate antigen which is Le y or LeX antigen or an antigen having the characteristics of Le y or LeX antigen, or a functional equivalent or fragment thereof, and a conjugate of a label which is a member of a signal producing system and a specific binding partner of the monoclonal antibody of above.

The diagnostic kit of Claim 19 wherein the label is an enzyme.

21. The diagnostic kit of Claim 19 wherein the label is a aa 2~ +j t r oil -27- fluorescer.

22. A diagnostic kit, comprising- a conjugate of a label which is a member of a signal producing system and a monoclonal antibody as defined in any one of Claims 1-8 useful for determining the presence of a malignant condition in the lung, said antibody defining a determinant site on a cell surface carbohydrate antigen having the characteristics of Ley or LeX antigen or a functional equivalent or fragment thereof, to

23. The diagnostic kit of Claim 22 wherein the label is a fluorescer.

24. The diagnostic kit of Claim 22 wherein the label is an enzyme.

A method for determining the presence of a malignant condition in human tissue, which comprises: contacting a specimen of said tissue with the monoclonal antibody as defined by Claim 1 under conditions for binding of said antibody to said specimen, and observing the presence of binding of said antibody to said specimen, said binding being related to the presence of a malignant condition in said tissue.

26. A method for determining the presence of a malignant condition in human tissue, which comprises: contacting a specimen of said tissue with the monoclonal antibody as defined by Claim 5 under conditions for binding of said antibody to said specimen, and observing the presence of binding of said antibody to said specimen, said binding being related to the presence of a malignant condition in said tissue.

27. A monoclonal antibody as claimed in Claim 1 substantially as hereinbefore described with reference to any one of the examples.

28. A monoclonal antibody as claimed in Claim substantially as hereinbefore described with reference to any one of the examples. Ir DATED: 3 July, 1990 INGEGERD HELLSTROM, KARL tRIX HELLSTROM, PETER LINSLEY, JOSEPH P. BROWN and DIANE HORN By their Patent Attorneys: PHILLIP'S ORI4ONDE FITZPATRICK JM04At/