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Title:
WT1 MONOCLONAL ANTIBODIES AND METHODS OF USE THEREFOR
Document Type and Number:
WIPO Patent Application WO/1995/029995
Kind Code:
A1
Abstract:
The present invention provides three unique monoclonal antibodies directed against a portion of the Wilms' tumor antigen, and methods of use therefor in detecting, monitoring and diagnosing malignancies characterized by over-expression or inappropriate expression of the WT1 protein.

Inventors:
HERLYN MEENHARD
MORRIS JENNIFER
RAUSCHER FRANK J III
RODECK ULRICH
Application Number:
PCT/US1995/005523
Publication Date:
November 09, 1995
Filing Date:
April 25, 1995
Export Citation:
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Assignee:
WISTAR INST (US)
International Classes:
C07K16/30; C12P21/08; (IPC1-7): C12N5/12; C07K16/18; C07K16/30; C12N15/02; C12P21/08; G01N33/574
Domestic Patent References:
WO1991007509A11991-05-30
Foreign References:
US5141736A1992-08-25
Other References:
ONCOGENE, Volume 6, Number 12, issued 12 December 1991, MORRIS et al., "Characterization of the Zinc Finger Protein Encoded by the WT1 Wilms' Tumor Locus", pages 2339-2348.
PROC. NATL. ACAD. SCI. U.S.A., Volume 90, issued June 1993, MAHESWARAN et al., "Physical and Functional Interaction Between WT1 and p53 Proteins", pages 5100-5104.
CLINICAL CHEMISTRY, Volume 27, Number 11, issued 1981, SEVIER et al., "Monoclonal Antibodies in Clinical Immunology", pages 1797-1806.
NATURE, Volume 351, issued 06 June 1991, CO et al., "Humanized Antibodies for Therapy", pages 501-502.
PROC. NATL. ACAD. SCI. U.S.A., Volume 81, issued November 1984, MORRISON et al., "Chimeric Human Antibody Molecules: Mouse Antigen-Binding Domains with Human Constant Region Domains", pages 6851-6855.
See also references of EP 0759071A4
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Claims:
WHAT IS CLAIMED IS:
1. A hybridoma cell line which produces a monoclonal antibody specific for an epitope located in Wilms' tumor protein antigen amino acids 1181 SEQ ID NO: 4.
2. The hybridoma cell line according to claim 1, wherein said cell line is ATCC No. 11598.
3. The hybridoma cell line according to claim 1, wherein said cell line is ATCC No. 11599.
4. The hybridoma cell line according to claim 1, wherein said cell line is ATCC No. 11560.
5. A monoclonal antibody directed against an epitope located in Wilms' tumor protein antigen amino acids 1181 SEQ ID NO: 4, said antibody capable of specifically binding to Wilms1 tumor protein.
6. The antibody according to claim 5, wherein said antibody is H2.
7. The antibody according to claim 5, wherein said antibody is H7.
8. The antibody according to claim 5, wherein said antibody is HC17.
9. A polypeptide derived from a monoclonal antibody directed against an epitope located in Wilms' tumor protein antigen amino acids 1181 SEQ ID NO: 4, said polypeptide selected from the group consisting of (a) heavy chain variable region polypeptides of said monoclonal antibody; (b) light chain variable region polypeptides of said monoclonal antibody; (c) a Fab fragment of said antibody; (d) a F(ab)2 fragment of said antibody; and (e) an Fv fragment of said antibody.
10. A method for diagnosing a disease condition characterized by WTl expression comprising the steps of: a) providing a biological sample from a patient having the clinical symptoms associated with mesothelioma; b) contacting said sample with a monoclonal antibody or functional fragment thereof specific for an epitope located in Wilms' tumor protein antigen amino acids 1181 SEQ ID NO: 4; and c) detecting the presence of binding of said monoclonal antibody or fragment to said biological sample, wherein the presence of such binding indicates the presence of said disease condition.
11. The method according to claim 10, wherein said antibody is selected from the group consisting of H2, H7, HC17, and a cocktail thereof.
12. The method according to claim 10, wherein said biological sample is selected from the group consisting of whole blood, serum, plasma, synovial fluid, and tissue and said disease condition is selected from the group consisting of mesothelioma, prostate cancer, ovarian cancer, and leukemia.
13. A method of monitoring therapy in leukemia patients comprising the steps of: a) providing a biological sample from a patient treated for leukemia; b) contacting said sample with a monoclonal antibody or functional fragment thereof specific for an epitope located in Wilms' tumor protein antigen amino acids 1181 SEQ ID NO: 4; and c) detecting the presence of binding of said monoclonal antibody or fragment thereof to said biological sample, wherein the presence of such binding indicates the presence of a active leukemia cells.
14. The method according to claim 13, wherein said antibody is selected from the group consisting of H2, H7, HC17, and a cocktail thereof.
15. The method according to claim 13, wherein said biological sample is selected from the group consisting of whole blood, plasma, serum, urine and bone marrow.
16. The use of a monoclonal antibody raised against an epitope located in Wilms' tumor protein antigen amino acids 1181 SEQ ID NO: 4 in detecting a disease characterized by the expression of the Wilms' tumor antigen.
17. A kit for diagnosing a disease characterized by the expression of the Wilms' tumor antigen comprising a monoclonal antibody raised against an epitope located in Wilms' tumor protein antigen amino acids 1181 SEQ ID NO: 4 and means for signal generation.
18. The kit according to claim 17 wherein said monoclonal antibody is selected from the group consisting of H2, H7 and HC17.
19. An antibody construct comprising at least one complementarity determining region from a monoclonal antibody specific for an epitope located in Wilms' tumor protein antigen amino acids 1181 SEQ ID NO: 4, said construct selected from the group consisting of a humanized antibody, a chimeric antibody, and a bi specific antibody.
20. The antibody according to claim 19 wherein said monoclonal antibody is selected from the group consisting of H2, H7 and HC17.
21. A method for producing an antibody construct comprising employing at least one complementarity determining region or heavy chain variable region from a monoclonal antibody specific for an epitope located in Wilms1 tumor protein antigen amino acids 1181 SEQ ID NO: 4.
Description:
WT1 MONOCLONAL ANTIBODIES AND METHODS OF USE THEREFOR

This work was performed with financial support under National Institutes of Health Grant Nos. CA25874, CA52009, CA47983 and CA10815. The United States Government has certain rights in this invention.

Field of the Invention

This invention relates generally to the field of detecting, monitoring and diagnosing malignancies characterized by expression of the Wilms 1 tumor 1 antigen.

Background of the Invention The Wilms' tumor (wtl) gene encodes a protein referred to as WTl which is expressed in the nucleus of certain cells and possesses the structural features of a DNA binding transcription factor. As illustrated in Fig. 1 below, the WTl protein is a 429 amino acid protein [SEQ ID NO:4] which contains four contiguous zinc fingers at the carboxyl-terminus, and a glutamine- and proline-rich region at the amino-terminus. The amino-terminal region of WTl protein mediates transcriptional suppression or activation in transient transfection assays [Madden et al, Science, 253:1550-1553 (1991); Maheswaran et al,

Proc. Natl. Acad. Sci. USA. £0.5100-5104 (1993); S. L. Madden et al, Oncogene, 8 :1713-1720 (1993)]. Splice variants of WTl can produce the protein with a 17 amino acid insert at amino acid 249 and/or a 3 amino acid insert at amino acid 390.

The wtl gene encoding WTl protein is located on chromosome llpl3 and has been found to be mutated or deleted in a subset of hereditary and sporadic Wilms' tumors. Recently, high levels of wtl expression were reported in a variety of tumors such as ovarian carcinomas [Bruenig et al, Cancer Invest. , 11:393-399

(1993)], prostate cancer, mesotheliomas [Park et al, cited above], and leukemias [Miwa et al, Leukemia- .6:405- 409 (1992), Miyagi et al, Leukemia. 7:970-977 (1992)]. Diagnostic methods for the ovarian carcinomas, mesotheliomas, and leukemias referred to above are based primarily on clinical attributes and histology of tumor specimens. These methods may at times not distinguish between closely related diseases and may lead to inappropriate treatments of patients. For example, in addition to the presence of many histological variants of malignant mesothelioma, there are other lesions that can affect the pleural surface and present a clinical and histological picture quite similar to malignant mesothelioma [R. J. Pisani et al, Mayo Clin. Proc. , .63:1234-1244 (1988)]. Additional relatively specific molecular markers that clearly distinguish between clinically similar lesions for malignant mesotheliomas as well as the other cancers would thus be a valuable clinico-pathological tool which will permit a precise diagnosis. This is important since treatment protocols and prognosis for such conditions vary significantly.

Currently available diagnostic tools include rabbit polyclonal antibodies for WTl protein known in the art. Morris et al, Oncogene, 6.2339-2348 (1991) describe two such antibodies which recognize amino acid fragments spanning amino acids 294-429 of SEQ ID NO:4 and amino acids 85-173 of SEQ ID NO:4, respectively, of the WTl protein. Another rabbit polyclonal antibody, which recognizes WTl amino acids 275-429 of SEQ ID NO: 4 was described by Telerman et al, Oncogene, 2:2545-2548 (1992) . Still other WTl polyclonal antibodies are commercially available, e.g. the rabbit polyclonal antibody SC-192, which is available from Santa Cruz. However, while polyclonal antibodies in general are able to detect WTl expression, they have disadvantages in

their potential for cross-reactivity with closely related proteins which share common domains with the WTl protein. These polyclonal antibodies by their nature are likely to provide inconsistent results in antigen specificity and binding affinity studies and are not particularly desirable for diagnostic uses.

Additionally, a commercially available mouse monoclonal antibody, DG-10 (Applied BioTechnology) was raised to the zinc finger region of WTl and is known to cross-react with the Egrl proteins. Expression of Egrl proteins is not limited to cells or tissues that express WTl and is independently regulated from WTl expression. Therefore, any antibodies raised to the zinc finger domain in the carboxyl terminus of WTl may not be useful for selective detection of the WTl.

Another anti-WTl mouse monoclonal antibody has been described by Mundlos et al, Development, 119:1329-1341 (1993) . The Mundlos et al antibody is specific for a 17 amino acid sequence insert (See Fig. 1 below), i.e., a splice variant, that is present in only a subpopulation of the alternatively spliced WTl mRNA messages.

Thus, there exists a need in the art for methods and compositions for detecting and differentially diagnosing conditions characterized by over-expression or inappropriate expression of WTl.

Summary of the Invention

In one aspect, the present invention provides a hybridoma cell line secreting a monoclonal antibody (MAb) specific for a protein antigen, referred to as WT1-6F

[SEQ ID NO: 2], which contains amino acids 1-181 of WTl [SEQ ID NO: 4]. One such cell line is an H2-secreting line, deposit designation ATCC No. 11598. Another cell line which is an embodiment of this aspect is the H7- secreting line, deposit designation ATCC No. 11599.

Still a third cell line is the HC17-secreting line, deposit designation ATCC No. 11600.

In another aspect, the present invention provides a monoclonal antibody produced by a cell line described above. Three such antibodies, designated H2, H7 and HC17 are described herein.

In yet another aspect, the invention provides the heavy chain and light chain variable region polypeptides of the MAbε of the invention, and other fragments thereof, such as Fab fragments, F(ab) 2 fragments, Fv fragments and the like.

In still another aspect, the present invention provides methods of diagnosing malignancies characterized by over-production or inappropriate expression of WTl protein. These methods involve screening biological samples with antibodies of the invention, described above.

In a further aspect, the present invention provides methods of monitoring treatment of conditions characterized by over-production or inappropriate expression or production of WTl protein. One embodiment of such a method involves monitoring leukemia treatment, particularly determining the level of active leukemia following a treatment cycle. In another aspect, the present invention provides methods for differentiating between malignancies characterized by over-production or inappropriate expression of WTl protein and conditions having similar symptomatic profiles. One embodiment of such a method involves distinguishing between mesotheliomas and conditions characterized by inflammatory reactions.

In a still another aspect, the present invention provides kits useful for detecting, monitoring, and/or diagnosing a disease characterized by the expression of the Wilms' tumor antigen comprising a MAb raised against

the WT-6F antigen [SEQ ID NO: 2]. Desirably, the H2, H7, HC17 MAbs or a cocktail of these, is included in such a kit.

Other aspects and advantages of the present invention are described further in the following detailed description of the preferred embodiments thereof.

Brief Description of the Drawings

Fig. 1 is a schematic diagram of the Wilms' tumor protein functional domains. The WTl protein contains two discrete functional domains: the amino terminus contains a transcriptional regulator domain and the carboxy terminus contains a DNA binding domain with four C 2 H 2 zinc fingers. G/P refers to the glutamine- and proline- rich region at the amino-terminus; ZN refers to four contiguous zinc fingers at the carboxyl-terminus. Alternatively spliced transcripts of WTl are produced which insert 17 amino acids, VAAGSSSSVKWTEGQSN, [SEQ ID NO: 7] (17AA) within the transcriptional regulatory domain (at amino acid 249 of SEQ ID NO: 4) or a tripeptide encoding the amino acid KTS within the DNA binding domain (at amino acid 390 of SEQ ID NO: 4) between zinc fingers 3 and 4. The significance of the alternatively spliced WTl transcripts is not known. Fig. 2 provides the nucleic acid and amino acid sequences of the WT-6F antigen [SEQ ID NOS: 1 and 2] in which amino acids 1-11 represent a histidine fusion protein to facilitate purification; amino acids 12-192 are amino acids 1-181 of the WTl protein; and amino acids 193-210 of SEQ ID NO: 2 are vector sequences added during cloning.

Fig. 3 provides the nucleotide and amino acid sequences of the full length WTl protein [SEQ ID NOS: 3 and 4] . The 3' non-coding sequence of the mRNA is omitted in this figure.

Detailed Description of the Invention

The present invention provides hybridomas secreting monoclonal antibodies (MAbs) specific for epitopes found in the amino terminal amino acids 1-181 of the Wilms* tumor (WTl) protein [SEQ ID NO: 4]. The MAbs of this invention are useful in identifying, monitoring and diagnosing conditions characterized by over-expression or inappropriate expression of the WTl protein. The MAbs do not cross-react with the ubiquitous and closely related early growth response (Egrl) family of proteins which share approximately 50% homology within the DNA binding domain located in the carboxyl terminal amino acids 275- 429 of WTl [SEQ ID NO:4]. Therefore, when used in a diagnosis based on the detection of WTl protein, the MAbs of this invention eliminate false positives currently produced in detection methods by the use of currently available WTl antibodies which are specific for epitopes in the zinc finger domain of the protein.

I. Definitions

As used herein "functional fragment" is a partial complementarity determining region (CDR) sequence or partial heavy or light chain variable sequence of an antibody which retains the same antigen binding specificity and/or neutralizing ability as the antibody from which the fragment was derived.

A "condition characterized by over-expression or inappropriate expression of WTl" refers to a cancer or other abnormal physiological state which exhibits an increased level of expression of WTl or exhibits expression of a mutant WTl protein, or exhibits expression of WTl protein where such expression should normally not occur. Such increased WTl expression has been detected in cells derived from ovarian carcinomas, mesotheliomas, prostate cancer and leukemias.

Ordinarily, in normal tissues, WTl protein is absent or present in such low levels that it cannot be detected using conventional techniques, such as northern blot hybridization or reverse transcriptase polymerase chain reaction (RT-PCR) . In contrast to WTl protein, when a patient exhibits a "condition characterized by over- expression or inappropriate expression of WTl" as defined herein, the presence of WTl protein can be detected using the reagents of the invention and standard techniques, e.g. immunohistoche ical procedures, including im unoblotting and immunofluorescence. Western blot analysis, and enzyme-linked immunosorbant assay (ELISA) . The presence of WTl mRNA in such patients can be detected using Northern blot analysis or RNA reverse transcription PCR techniques. Background levels of WTl can be determined by measuring such levels in the tissues where WTl is not normally expressed (as described above) in persons not afflicted with disease.

"CDRs" are defined as the complementarity determining region amino acid sequences of an antibody. CDRs are contained within the hypervariable regions of i munoglobulin heavy and light chains. CDRs provide the majority of contact residues for the binding of the antibody to the antigen or epitope. CDRs of interest in this invention are derived from donor antibody variable heavy and light chain sequences, and include functional fragments and analogs of the naturally occurring CDRs, which fragments and analogs also share or retain the same antigen binding specificity and/or neutralizing ability as the donor antibody from which they were derived. By 'sharing the antigen binding specificity or neutralizing ability' is meant, for example, that although a given MAb may be characterized by a certain level of antigen affinity, and a CDR encoded by a nucleic acid sequence of the same MAb in an appropriate

structural environment may have a lower or higher affinity, it is expected that CDRs of that MAb in such environments will nevertheless recognize the same epitope(s) as the MAb from which they are derived. A "monoclonal antibody" refers to homogenous populations of immunoglobulins which are capable of specifically binding to WTl protein. It is understood that WTl protein may have one or more antigenic determinants, particularly in the amino acid sequence spanning amino acids 1-181 of SEQ ID NO: 4. The antibodies of the invention may be directed against one or more of these determinants. As used herein, a "cocktail" of these antibodies comprises any combination of the antibodies of the invention. A "chimeric antibody" refers to a type of engineered or recombinantly produced antibody which contains naturally-occurring variable region light chain and heavy chains (both CDR and framework regions) derived from a non-human donor antibody, such as the MAbs described herein, in association with light and heavy chain constant regions derived from a human (or other heterologous animal) acceptor antibody.

A "humanized antibody" refers to a recombinantly produced antibody having its CDRs and/or other portions of its light and/or heavy variable domain framework regions derived from a non-human donor immunoglobulin, such as the MAbs of the present invention, the remaining immunoglobulin-derived parts of the molecule being derived from one or more human immunoglobulins. Such antibodies can also include a humanized heavy chain associated with a donor or acceptor unmodified light chain or a chimeric light chain, or vice versa.

A "bi-specific antibody" refers to an antibody derived from the Fab portions of two parent antibodies, each of which binds a separate antigen. The bi-specific

antibody is characterized by the ability to bind to two antigens, particularly, the antigens to which the parent antibodies bound.

A Fab fragment refers to a polypeptide containing one entire light chain and amino terminal portion of one heavy chain from an antibody, such as the MAbs of this invention. A F(ab*) 2 fragment refers to the fragment formed by two Fab fragments bound by disulfide bonds.

II. Hybridoma Cell Lines and MAbs of the Invention The hybridoma cell lines and monoclonal antibodies of the invention are produced by employing as antigen, a novel WTl-derived protein antigen, which contains only the N-terminal sequence of the WTl protein. Desirably, the invention employs as an immunogen a WTl containing protein antigen, referred to as WT1-6F [SEQ ID NO: 2], which contains amino acids 1-181 of the N-terminus of the native human WTl sequence (see Fig. 1 and SEQ ID NO: 4) . This antigen has been developed by the inventors and does not contain any of the zinc-finger region characteristic of the carboxyl terminal portion of the WTl protein or any of the 17 amino acid insert of the splice variant of the protein (see Fig. 1) . Additional details relating to the preparation and expression of the 6F antigen are provided in Example 1 below.

Generally, the hybridoma process involves generating a B-lymphocyte to the selected antigen, which B lymphocyte produces a desired antibody. Techniques for obtaining the appropriate lymphocytes from mammals injected with the target antigen, WT1-6F, are well known. Generally, the peripheral blood lymphocytes (PBLs) are used if cells of human origin are desired. If non-human sources are desired, spleen cells or lymph nodes from other mammalian sources are used. A host animal, e.g. a mouse, is injected with repeated doses of the purified

antigen, and the mammal is permitted to generate the desired antibody producing cells.

Thereafter the B-lymphocytes are harvested for fusion with the immortalizing cell line. Immortalizing cell lines are usually transformed mammalian cells, particularly cells of rodent, bovine and human origin. Most frequently, rat or mouse myeloma cells are employed. Techniques for fusion are also well known in the art and generally involve mixing the cells with a fusing agents, e.g. polyethylene glycol.

Immortalized hybridoma cell lines are selected by standard procedures, such as HAT selection. From among these hybridomas, those secreting the desired monoclonal antibody are selected by assaying the culture medium by standard immunoassays, such as Western blotting, ELISA, or RIA. Antibodies are recovered from the medium using standard purification techniques. See, generally, Sambrook et al, Molecular Cloning: A Laboratory Manual. 2nd edit., Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1989) . Alternatively, non-fusion techniques for generating an immortal antibody-producing hybridoma cell line may be employed to generate a hybridoma antibody, where possible, e.g. virally induced transformation. The invention provides three exemplary hybridoma cell lines and the MAbs secreted therefrom produced using WT1-6F as the antigen. See Examples 2 and 3 below. These three hybridomas secrete antibodies termed H2, H7 and HC17, respectively. Each hybridoma was deposited with the American Type Culture Collection, 12301 Parklawn Drive, Rockville, Maryland, U.S.A. ("ATCC") on March 31, 1994, pursuant to the provisions of the Budapest Treaty. The H2-secreting hybridoma was granted accession number ATCC 11598, the H7-secreting hybridoma was granted

accession number ATCC 11599, and the HC17-secreting hybridoma was granted accession number ATCC 11600.

The H2, H7 and HC17 antibodies are murine IgG 1 antibodies, and have been demonstrated to specifically bind WTl protein and not to cross-react with the closely- related Egrl family of proteins. All three monoclonal antibodies recognize the recombinant protein in ELISA assays, and full length WTl protein in immuno- precipitation and Western blot analysis. Preliminary analysis suggests that at least two distinct epitopes in the WT1-6F protein are recognized by the three MAbs. The MAbs of this invention are characterized in more detail in Example 4 below.

A Western blot analysis was performed to test the ability of the three MAbs to detect two recombinant proteins: 6F [SEQ ID NO: 2] which contains WTl amino acids 1-181 of SEQ ID NO: 4, and WT91 which contains WTl amino acids 85-173 of SEQ ID NO: 4. All three MAbs detect the 6F recombinant protein [SEQ ID NO: 2] containing WTl amino acids 1-181. However, only H2 and H7 detect the WT91 recombinant protein containing amino acids 85-173 of WTl, suggesting that H2 and H7 recognize an epitope within the WTl amino acid sequence 85-173 and HC17 recognizes an epitope outside this region. These MAbs are useful as diagnostic reagents, and possibly as therapeutic reagents as described in more detail below.

III. MAb Antibody Fragments The present invention also includes functional fragments of the MAbs defined above, preferably those derived from the H2, H7 and/or HC17 MAbs of the invention. Such functional fragments include the heavy chain and light chain variable region polypeptides of the

MAbs of the invention, and other fragments thereof, such as Fab fragments, F(ab) 2 fragments, Fv fragments and the like.

These fragments are useful as diagnostic reagents and as donors of sequences, including the variable regions and CDR sequences, useful in the formation of recombinant, chimeric, humanized or bi-specific antibodies. Techniques for generating such antibodies and antibody fragments are known in the art. For example, the functional fragments of the invention may be obtained using conventional genetic engineering techniques. See, generally, Sambrook et al., cited above. Alternatively, desired portions thereof, e.g. the CDR sequences, may be chemically synthesized. These antibody functional fragments are useful in the assays of the invention to diagnose WTl over- expression or inappropriate expression in specific tumors, which assays are described in more detail below. For example, by conjugating these antibody fragments to enzymes, such as horseradish peroxidase, these fragments may be employed in a conventional one-step detection assay.

IV. Diagnostic Reagents and Kits The invention includes kits of reagents for use in i munoassays, particularly sandwich immunoassays. Such kits include a solid phase support, a monoclonal antibody of the invention, a functional fragment thereof, or a cocktail thereof, and means for signal generation. The antibodies of the invention may be pre-attached to the solid support, or may be applied to the surface of the solid support when the kit is used. The signal generating means may come pre-associated with an antibody of the invention or may require combination with one or more components, e.g. buffers, antibody-enzyme

conjugates, enzyme substrates, or the like, prior to use. Kits may also include additional reagents, e.g. blocking reagents for reducing nonspecific binding to the solid phase surface, washing reagents, enzyme substrates, and the like. The solid phase surface may be in the form of microtiter plates, microspheres, or other materials suitable for immobilizing proteins. Preferably, an enzyme which catalyzes the formation of a chemiluminescent or colored product is a component of the signal generating means. Such enzymes are well known in the art.

Such kits are useful in the detection, monitoring and diagnosis of conditions characterized by over- expression or inappropriate expression of the WTl protein.

V. Diagnostic Assays

The MAbs, fragments, reagents and kits of the invention may be used with standard assay formats for the identification and diagnosis of conditions characterized by WTl expression, over-expression or inappropriate expression, particularly in tumor/leukemic cells. The detection and measurement of WTl expression in tissue that does not normally express WTl or over-expression or inappropriate expression in tissue that does normally express WTl may be accomplished by resort to several known techniques, e.g., immunofluorescence (detection of WTl protein in fixed cells/tissues) and detection of WTl protein of whole cell extracts by western analysis. Most particularly, the MAbs and other compositions of this invention may be used to detect WTl expression in abnormal kidney and genitourinary development and cancers which over-express WTl, particularly, leukemias, mesothelioma, granulosoma, prostate and ovarian cancers.

The reagents of the invention may also be used to monitor therapy of such conditions.

Desirably, the MAbs and fragments thereof, when used as diagnostic reagents are conventionally labelled for use as molecular weight markers or for use in ELISAs, immunofluorescence, and other conventional assay formats for the measurement of WTl in an appropriate biological sample. Suitable label systems are well known to those of skill in the art and include fluorescent compounds, radioactive compounds or elements, and a variety of enzyme systems. As used herein, suitable samples include, without limitation, whole blood, serum, plasma, tissue samples, bone marrow, and urine.

Advantageously, the MAbs of the invention can be used to screen for the WTl protein using standard antibody staining techniques, e.g. the avidin-biotin system, immunofluorescence, or the like. For example, a tissue, e.g. from a biopsy, is fixed on a slide using standard techniques. A selected MAb (or fragment thereof) of the invention is then applied to the slide and incubated under standard conditions, e.g. at room temperature for about 1 hour. A labelled anti-mouse antibody is then used for detection. Parallel experiments with positive and negative controls (minus MAb of invention) are performed.

Significantly, if the MAbs of the invention avoid interference with MAb recognition by fixation of the tumor tissue with conventional reagents, e.g. paraformaldehyde and, preferably, methanol, these antibodies may be useful on routine pathology slides.

For example, the ability of these monoclonal antibodies to detect prostate cancer cells has been demonstrated. Preliminary data has demonstrated that cocktails of these antibodies, e.g., H2/HC17 and H7/HC17, are particularly well suited for this purpose.

The MAbs, or functional fragments thereof, of the invention are useful in the detection of a condition characterized by over-expression of WTl antigen, including leukemias, mesothelioma, and granulosoma, or to differentiate such a condition from other conditions which exhibit similar clinical symptoms. For example, a Mab of the invention can differentiate a mesothelioma from other pleural tumors; such a use is clinically significant in view of the different prognoses for pleural tumors of non-adenocarcinoma origin and adenocarcinomas. Such a method involves obtaining a suitable biological sample from a patient, incubating the sample in the presence of a Mab or functional fragment thereof of the invention, and detecting the presence of binding of the Mab or fragment to the biological sample. The presence of binding above background levels detected in a non-WTl expressing normal tissue sample indicates the presence of a mesothelioma. Any tissue or established cell line which does not express WTl MRNA may serve as a standard for negative expression of WTl protein, including those described above in the background.

Alternatively, the Mabs and fragments thereof of the invention are useful to monitor a course of treatment for a condition characterized by over-expression or inappropriate expression of the WTl antigen. For example, active leukemia (e.g. in blast crisis) cells express WTl, while inactive leukemic cells do not express WTl. Thus, during or following a treatment cycle, a biological sample from the leukemia patient is periodically tested in an assay of the invention to monitor residual leukemic disease. The lack of, or reduction of levels of, binding of a Mab or fragment of the invention to the sample indicates that the course of treatment, e.g., chemotherapy, is successful in reducing

the tumor or cancer. Similarly, the MAbs and fragments of the invention may be used to detect leukemic blast cells in purged or unpurged hematopoietic stem cell preparations intended for use in bone marrow transplantation.

It is anticipated that one of skill in the art of diagnostic assays may devise other series of steps utilizing the Mabs or fragments of this invention to accomplish the detection of levels of WTl expression indicative of disease. Such assay formats are known within the art, and are simply a matter of selection. This invention is not limited by the particular assay format or assay steps employed in the diagnosis of inappropriate expression of WTl protein in biological samples.

Because the Mabs H2, H7, and HC17 were raised to a region of the WTl amino acid sequence that is unique to the amino terminal portion of WTl and does not contain the zinc finger DNA binding domains, these Mabs and fragments have little potential for crossreactivity with non-WTl proteins, unlike known other WTl polyclonal and monoclonal Mabs. For example, these Mabs do not cross- react with the Egr family of proteins. Thus they permit an unambiguous positive detection of WTl expression in biological samples.

The advantages of using these Mabs for such diagnosis in comparison to the use of the known monoclonal and polyclonal antibodies of the art rely in the defined specificity of the Mabs for the amino terminal sequence of WTl, their uniform binding affinity and their lack of cross-reactivity as described above.

V. Therapeutic Use of Mabs of this Invention

Further, if these Mabs of the invention are have the ability to internalize into the nucleus of the cell which expresses WTl [see, e.g., United States Patent No. 5,296,348, issued March 22, 1994, incorporated by reference herein] , they may also be employed in the treatment of such WT-1 expressing tumors or cancers. For example, these Mabs, other antibody types such as chimeric or humanized antibodies, or fragments which share the binding affinity or specificity of the whole Mab may be used to deliver toxins or therapeutic agents to the tumor or metastasis sites.

These Mabs, other antibodies and fragments of the present invention may also be employed in other therapeutic methods known to those of skill in the art. The following examples illustrate the characterization and uses of the antibodies of the invention. These examples are illustrative only and do not limit the scope of the invention.

Example 1 - Preparation of the WT1-6F Antigen A. Cloning Strategy

A recombinant protein containing the first 181 amino acids of the human WTl was produced to use as an antigen in the preparation of WTl specific antibodies as follows.

The amino terminus of WTl was subcloned from 7Zf+WTl, a synthetic full-length human WTl nucleotide sequence described in Morris et al, cited above. Briefly, the nucleotide sequence encoding the full-length protein was: constructed from the partial human WTl cDNA clone WT33 [Call et al, Cell. 60:509-520 (1990)]. The WTl amino acids 1-84 plus an overlapping 20 amino acid segment were synthesized using overlap-extension

polymerase chain reaction. The nucleotides specifying amino acid codons were optimized for expression in E. coli .

The resulting synthetic DNA fragment (320 bp) was fused to the 5' end of WT33 at a unique Bst XI site centered at position WTl amino acid 101 of SEQ ID NO: 4 (nucleotide 50 of WT33) . A Cla I-Eco RI fragment was subcloned into pGem7Zf+ (Promega, Madison, WI) to produce 7Zf+WTl. From this plas id, a Nco I-Bam HI fragment was isolated and subcloned into a modified petlld vector (Novagen, Madison, WI) .

The petlld vector was modified by digesting with Nco I and ligating to synthetic, double-stranded oligonucleotides which contained the following 5' overhangs complementary to a Nco I restriction site to produce 6XHISpetlld:

5 '-CATGAGAGGATCGCATCACCATCACCATCACTC 3 '[SEQ ID NO: 5] 3* TCTCCTAGCGTAGTGGTAGTGGTAGTGAGGTAC-5' [SEQ ID NO: 6]. The synthetic oligonucleotide introduces nucleotide codons that encode the amino acids MRSHHHHHH of SEQ ID NO: 2 to produce an in-frame 5' hexa-histidine fusion protein that maintains a single Nco I restriction site at the 3' end of the sequence. The 5' hexa- histidine encoding region facilitates protein purification [Abate et al, Proc. Natl. Acad. Sci. USA. 87 . :1032 (1990)].

The Nco I-Bam HI fragment of 7Zf+WTl containing the amino terminus of WTl was subcloned into 6XHISpetlld digested with Nco-I and Bam HI to create petlld-6F. B. Expression in E. coli and Purification

The bacterial strain BL21 (Novagen, Madison, WI) was transformed with the petlld-6F DNA. Protein expression was induced in a logarithmically growing bacterial culture with 1 mM isopropyl-β-thiogalacto- pyranoside (IPTG) for two to three hours at 37°C.

Bacteria were harvested by centrifugation, lysed in 6 M guanidine-HCl, 50 mM sodium phosphate, pH 8.0 for 2 hours at room temperature or overnight at 4°C, and clarified by centrifugation at 40,000 x g for 30 minutes. The histidine fusion recombinant protein WT1-6F was purified by a one step affinity binding of the hexa- histidines to the nickel-chelate affinity resin NTA- agarose (Qiagen, Chatsworth, CA) using recommended procedures. The purified protein was renatured by dialysis into phosphate buffered saline with 5% glycerol. Purity of the protein was determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS- PAGE) as follows. The purified protein was renatured by dialysis into PBS containing 5% glycerol and analyzed on a 10% SDS polyacrylamide gel. Proteins were stained with Coomassie blue. The 6F recombinant protein was shown to be homogenous, migrating under denaturing conditions as a 28 kDa protein.

C. The WT1-6F Antigen The 6F amino acid sequence is encoded by a synthetic nucleotide sequence shown in Fig. 2 [SEQ ID NOS: 2 and 1] . The 6F nucleotide sequence was derived from the synthetic full-length human WTl sequence [Morris et al, cited above and SEQ ID NOS: 3 and 4]. As illustrated in Fig. 2, the recombinant 6F antigen contains amino acids 1-181 of the human WTl sequence [SEQ ID NO:4] as well as amino acids at both the amino and carboxyl ends, which sequences were introduced during cloning. The entire 6F amino acid sequence is shown in Fig. 2 [SEQ ID NO: 2]. Amino acids 1-11 (MRGSHHHHHHS) of SEQ ID NO: 2 were added to produce a histidine fusion protein to facilitate purification of the recombinant protein. Amino acids 193-210 of SEQ ID NO: 2 are not WTl sequences, but were added from the vector during cloning. Note that nucleotides 1-333 [SEQ ID NO: 1] are synthetic

sequences optimized for protein translation in E . coli ; the remaining nucleotides are derived from the human WT33 cDNA clone. This does not change the human WTl amino acid sequence, but increases efficiency of protein expression in I5__ coli [Rauscher et al, Science. 250:1259- 1262 (1990) , Abate et al, Proc. Natl. Acad. Sci.. 82:1032-1036 (Feb. 1990)].

A second recombinant protein, WT91 (described in Morris et al, cited above) contains the amino acids 85-173 of SEQ ID NO: 4.

Example 2 - Preparation of Antisera and Immunization

Rabbit polyclonal antisera was produced by CoCalico Biologicals, Inc. Rabbits were immunized subcutaneously with 100 μg of 6F recombinant protein of Example 1 and boosted at two to three week intervals. The rabbit sera was used without further purification.

Example 3 - Preparation of Monoclonal Antibodies Fifty icrograms of purified recombinant protein of Example 1 was injected subcutaneously into the hind footpads of BALB/c mice every two weeks for a total of three injections. Sera was collected from the tail, and tested for WTl specific antibodies by immuno- precipitation of 35S-methionine labeled in vitro translated human WTl protein.

Two weeks later, 50 μg of protein in 200 μl of saline was injected intravenously followed by removal of each animal's spleen. Spleen cells were fused with a myeloma cell line, P3X63AG8/653 [ATCC CRL 1580], using standard techniques.

The resulting hybridomas producing MAbs H2, H7 and HC17 were screened in a two step process. Positive clones were initially identified using an enzyme-linked immunosorbent assay (ELISA) against the 6F recombinant

protein. Secondary screening was carried out using immunoprecipitation of full length WTl protein produced by in vitro translation (IVT) . These experiments demonstrated that the MAbs H2, H7 and HC17 specifically recognize the WTl protein and that they appear to recognize distinct epitopes on the WTl protein. 1. Immunoprecipitation

Full length WTl was expressed in vitro from by transcribing RNA from Eco RI linearized vector 7Zf+WTl with SP6 RNA polymerase, and translating protein in rabbit reticulocyte lysate with 35 S-methionine. The 35 S- methionine labeled protein is 55 kDa and is specifically immunoprecipitated by rabbit polyclonal anti-6F sera, and by the mouse monoclonal antibodies H2, H7, and HC17. Immunoprecipitations were done as previously described in Morris et al, cited above. Briefly, IVT WTl was added to radioimmunoprecipitation buffer with protease inhibitors (RIPA: 10 mM Tris-Cl pH 7.4, 150 mM sodium chloride, 1 mM ethylenediamine-tetraacetic acid (EDTA) , 1% Triton X-100, 1% deoxycholate, 0.1% SDS, 0.1 mM phenylmethylsulfonic acid (PMSF) , 2 μg/ml leupeptin and 2 μg/ml aprotinin) along with antibodies and incubated 90 minutes at 4°C. Either 30 μl of 10% Staphylococcus A (Pansorbin, Calbiochem, San Diego, CA) or 100 μl of 50% Protein A Sepharose (Pharmacia,

Piscataway, NJ) was added and incubated for 15 minutes (Staph A) or 30 minutes (Protein A) . The immune complexes were collected by centrifugation in the microfuge and washed with 0.5-1.0 ml of RIPA 3 to 4 times. The. immunoprecipitated proteins were analyzed on 10 or 15% SDS-polyacrylamide gels and visualized by autoradiography.

The resulting SDS PAGE gel demonstrated that MAbs of this invention immunoprecipitate WTl expressed by in vitro transcription and translation.

2. Baculovirus expression of full length WTl The full length WTl protein encoding sequence was subcloned from 7Zf+WTl into a baculovirus expression vector. Sf9 insect cells were infected with WT1- baculovirus and cells harvested 48-96 hours following infection. Cells were pelleted by centrifugation, washed three times in PBS. Whole cell lysates were prepared by lysing a cell pellet in 10 times the cell pellet volume with Laemmli loading buffer (50 mM Tris-Cl, pH 6.8, 100 mM dithiothreitol, 2% SDS, 0.1% bromophenol blue, 10% glycerol) .

Ten μl of WTl lysate were analyzed on a 10% SDS-polyacrylamide gel. Western analysis of protein was performed as follows. A whole cell lysate of Sf9 cells expressing baculovirus encoded WTl protein was separated on a 10% SDS-polyacrylamide gel and transferred to 0.45 μm BA 85 nitrocellulose (Schleicher and Schuell, Keene, NH) using semi-dry electroblot transfer for 60-90 minutes at 4 mAmps/cm 2 . Molecular weight standards were cut from the blot and stained with A ido black and the nitrocellulose filter blot was blocked in 5% BSA-PBS for 60 minutes at room temperature or overnight at 4°C. The primary antibody was diluted in blocking buffer (rabbit anti-6F 1:400; the monoclonal antibodies of the invention 1:500 or 1:1000) and added to filters for 30 to 60 minutes at room temperature.

Filters were rinsed briefly twice in wash buffer (PBS, 0.1% BSA, 1% Tween 20) and three times for 10 minutes each while shaking vigorously. Soluble protein A conjugated to horseradish peroxidase (Amersham, Arlington Heights, IL) was diluted 1:5000 in 5% BSA-PBS and incubated for 30 minutes at room temperature. Filters were washed as before, rinsed in PBS, and incubated with a 1:1 mixture of the ECL substrates A and B (Amersham, Arlington Heights, IL) for 1 minute at room

temperature. Filters were removed from the liquid, excess moisture drained, and wrapped in Saran wrap and immediately exposed to film (average exposure 15 seconds to 3 minutes) .

The gels revealed that the polyclonal and monoclonal antibodies of this invention specifically detect a 55 kDa protein in Sf9 cells transfected with WTl baculovirus expression vector and not cells mock transfected.

Example 4 - Characterization of Murine MAbs H2. H7 and HC17

To determine whether the WTl monoclonal antibodies of the present invention detect different epitopes within the first 181 amino acid of the 6F antigen, purified recombinant proteins 6F (WTl amino acid 1-181) and WT91 (WTl amino acid 85-173) were separated on a 15% SDS- polyacrylamide gel and transferred to nitrocellulose. Western blot analysis was performed as described in Example 3.

Polyclonal antibodies were diluted 1:400 and monoclonal antibodies diluted 1:500. The polyclonal antisera recognizes both the 6F and WT91 recombinant proteins. The monoclonal antibodies H2 and H7 recognize both 6F and WT91 recombinant proteins, suggesting they detect an epitope with amino acid 85-173 of WTl [SEQ ID NO:4]. HC17 does not detect the WT91 recombinant protein indicating that it recognizes an epitope outside of this region.

Example 5 - Detection of WTl Protein in Human Acute Leukemias

The following study demonstrates that a MAb of the invention, H2, is able to distinguish between leukemic blast cells and normal mononuclear cells by detecting the WTl protein in nuclei of leukemic blast cells. No WTl protein was detected in the nuclei of normal mononuclear cells or mononuclear cells by either immunofluorescence or by conventional reverse-transcriptase polymerase chain reaction (RT-PCR) techniques. A. Samples

Mononuclear cell preparations of 110 adult leukemia patients were examined in this study, T-cell acute lymphoblastic leukemias (T-ALL) had been diagnosed in 27, common acute lymphoblastic leukemias (c-ALL) in 28, pre-pre-B cell acute lymphoblastic leukemias (ppB- ALL) in 8, acute myelogenous leukemias (AML) in 40, chronic myelogenous leukemias in blast crisis (one lymphatic and three myeloid CML-BC) in 4 and chronic myelogenous leukemias in chronic phase (CML-CP) in 3 patients. Controls were 4 patients with reactive bone marrow aspirates who had fever of unknown origin (H.M. , G.S.), anemia secondary to iron deficiency (V.H.) or limited-disease esophageal cancer with no morphological evidence of bone marrow infiltration (H.F.).

Mononuclear cells were isolated from bone marrow aspirates or in a few cases from peripheral blood samples by Ficoll-Hypaque density gradient centrifugation (Pharmacia, Freiburg, Germany) . Also, peripheral mononuclear cells enriched with CD34 + hematopoietic progenitors were obtained from five patients (S.K., S.Kt., K.D., N.G., H.G.) who had solid cancer with no morphological evidence of bone marrow infiltration. Their mononuclear cells had been harvested by leukaphereεis during the recovery phase following a

course of progenitor-cell-mobilizing chemotherapy and G- CSF. Furthermore, a 91% pure peripheral CD34 + hematopoietic progenitor cell suspension was prepared from the leukapheresis product of a patient (G.M.) suffering from plasmacytoma.

The number of peripheral CD34 + progenitors was determined using a FACScan cytofluorometer. At least 10 5 CD34 + vital cells per sample were available for testing. In addition, nucleated blood cells of twenty patients with non-neoplastic disease were isolated using a red blood cell lysis-buffer (150 mM NH 4 C1, 10 mM KHC0 3 , and 0.1 mM EDTA) . The leukemia cell line K562 [ATCC CCL 243] served as the positive control in detection of wtl mRNA and in immunofluorescence studies. B. Indirect Immunofluorescence Assay

For the indirect immunofluorescence assay, mononuclear cells of bone marrow were isolated as already described. In addition, a 91% pure CD34 + hematopoietic progenitor cell suspension was prepared from the leukapheresis product of a patient (G.M.) suffering from plasmacytoma. Prior to leukapheresis, she underwent peripheral stem-cell mobilization with chemotherapy (VAD- protocol) and G-CSF.

An aliquot taken from the leukapheresis product contained 2.5 x 10 8 mononuclear cells and, according to FACS analysis [M. Notter et al, Blood. 82.3113 (1993)], 8.75 x 10 6 CD34 + hematopoietic progenitor cells. First, T-lymphocytes and myeloid cells were depleted using paramagnetic microbeads coupled with mouse anti-human CD3 and CD33 MAbs (Miltenyi, Cologne, Germany) . Using a B2 column (Miltenyi) , the cells were sorted according to the manufacturer's instructions. Subsequently, CD34 + hematopoietic progenitor cells were isolated using the CD34 Progenitor Isolation Kit (QBEND/10) from Miltenyi. After removal of unbound MAb by washing, cells were

passed twice over a Mini MACS column (without flow resistor, Miltenyi) . The 8G12-PE MAb (Becton Dickinson, Heidelberg, Germany) was used to determine the purity of the final CD34 + cell suspension, which was 91% with a yield of 39%.

One fraction of the cell preparations was processed according to the RT-PCR protocol described to detect the wtl transcript. Another fraction was used in the immunofluorescence assay. K562 cells served as positive controls. For detection of the nuclear protein WTl, 5 x 10 4 mononuclear cells were cytocentrifuged onto glass slides and air-dried for 2 hours. To destroy cellular membranes, the cells were exposed to pure methanol for 30 minutes at 4°C and then washed twice in PBS. The cells were incubated for 45 minutes at 4°C with the mouse antihuman WTl MAb H2, produced as described in Example 3 above, or a negative control MAb (MAb 425) recognizing the EGF-receptor [Rodeck et al, Cancer Res.. 42:3692 (1987)]. The cells were washed again in PBS and incubated for 30 minutes with fluoresceinisothiocyanate (FITC)-conjugated goat antimouse F(ab') 2 fragments (Immunotech, Marseille, France) . After washing in PBS, cells were embedded in PBS-glycerin and analyzed by fluorescence microscopy (Axiophot, lOOOx, Zeiss, Oberkochem, Germany) . Results are reported below in Table 1.

Table 1

Patient Wtl mRNA Nuclear Immunofluorescence

Diagnosis Initials Expression MAb H2 MAb 425

ALL pre-pre-B-ALL C.R. yes #yes $no c-ALL R.P. yes yes no c-ALL F.G. yes yes no c-ALL W.T. no no no

T-ALL A.D. yes no no

T-ALL M.S. no no no

AML

AML-FAB-M2 M.E. yes yes no

AML-FAB-M4 A.M. yes yes no

AML-FAB-M2 H.K. yes no no

AML-FAB-M1 H.L. no no no

Controls

K562 cells yes yes no

CD34+91% pure progenitor cells G.M. no no no normal blood mononuclear cells no no no

# indicates more than 30% of cells show a strong nuclear fluorescence. $ indicates no cells show nuclear fluorescence.

The indirect immunofluorescence assay with the MAb H2 directed against the WTl nuclear protein disclosed a strong and specific nuclear fluorescence in blast cells from 3 of 6 ALL patients and 2 of 4 AML patients tested (Table 1) . No nuclear immunofluorescence was observed in 3 ALL patients, one with (A.D.) and two without wtl gene expression. In mononuclear cell preparations from 4 AML patients a nuclear immunofluorescence with MAb H2 was found in 2 cases and both tested positive for wtl mRNA expression using RT-PCR. While blast cells of one AML patient did not express the wtl mRNA and had no nuclear immunofluorescence with MAb H2, those of another AML patient did show transcription of the wtl mRNA but no nuclear immunofluorescence (H.K. , Table 1) . K562 cells

showed strong nuclear immunofluorescence with MAb 6F-H2, whereas normal mononuclear blood cells and cells of a 91% pure CD34 + hematopoietic progenitor cell suspension did not (Table 1) . There was no nuclear immunofluorescence detectable using the negative control MAb 425 (Table 1) . In normal blood granulocytes, cytoplasmic but no nuclear fluorescence was found with MAb H2 and regarded as unspecific (data not shown) .

Iinmunofluorescence using MAb H2 confirms RT-PCR data, and shows detection of the WTl protein in nuclei of leukemic blast cells but not in those of normal mononuclear cells or mononuclear cells enriched with CD34 + hematopoietic progenitors.

Expression of protein occurs following the transcription of mRNA message from the double stranded

DNA. This mRNA is translated into a protein. Detectable mRNA indicates that the necessary "intermediate" is present and potentially capable of being translated into protein. However, this correlation does not always occur and the presence of mRNA does not necessarily mean the protein is being produced. Therefore, immunofluorescence detects protein expression and is the preferable assay system.

Example 6 - Detection of WTl Protein in Malignant Mesotheliomas

A. Cell Lines

The mesothelioma cell lines (ML1-ML19) used in the study were all developed from human mesothelioma tumors diagnosed using conventional immunohistochemical tests. Cell lines ML-10 and ML-16 were established by explant culture at the University of Pennsylvania [W. R. Smythe et al, Ann. Thorac. Surg.. 52(6) :1395-1401 (1994)]. Both cell lines have been passaged over 25 times without evidence of senescence, grow as tumors in

immunodeficient mice, and show a staining pattern characteristic of mesothelioma with lack of staining with LeuMl and carcinoembryonic antigen (CEA) antibodies. Cell lines ML1-ML8 were developed in the Surgical Oncology Laboratory at the National Cancer Institute (USA) . Mesothelioma cell lines, (ML11-ML15) and lung cancer lines (LL5-LL8) were provided by Dr. Carmen Allegra from the Medical Oncology Branch, NCI-Navy, National Naval Medical Center. Cell lines: ML9 (H-Meso) , ML17, ML18 and ML19 were provided by Dr. Joseph Testa from Fox Chase Cancer Institute, Philadelphia, PA. Normal mesothelial cells were developed from explants derived from non-malignant visceral pleural tissue obtained at surgery. These cell lines were maintained in RPMI-40 media (Gibco-BRL, Gaithersburg, MD) supplemented with 10% fetal calf bovine serum, non-essential amino acids (10 mM) , L-Glutamine (200 mM) , penicillin (0.1 mg/ml) and streptomycin (0.1 mg/ml). The six lung cancer cell lines, LL1 (A549) , LL2 (Calu-1) , LL3 (Calu-3) , LL4 (Calu- 6) , LL9 (SK-LU-1) , LL10 (SK-MES-1) . were purchased from American Type Culture Collection (ATCC) and cultured per instructions. Normal bronchial epithelial cells [S. A. Mette et al. Am. J. Respir. Cell. Mol. Biol.. 8.:562-572 (1993)] (HBE4) and human umbilical vein endothelial cells were cultured as described in S. M. Albelda et al, J. Clin. Invest.. 82:1992-2002 (1989)]. B. Transfection Protocol

To generate a positive control for cellular localization studies of WTl protein, COS-1 cells (ATCC) were either seeded at 5 x 10 4 cells/cm 2 onto 1% gelatin- coated coverslips or at 5 x 10 5 cells in a 35 mm dish and maintained in DMEM (Gibco-BRL, Gaithesburg, MD) plus 10% fetal bovine serum. Twenty-four hours later, 2.5 μg of pCMVhuWTlcDNA, an expression vector described previously

[Morris et al, cited above] was transfected into the cells by the calcium phosphate-mediated co-precipitation method [J. Sambrook et al, cited above]. Three days later the cells on the coverslips were processed for immunofluorescence staining with WTl antibody and cells in 35 mm dish were harvested for im unoblot analysis which is described below.

C. Human Tissue and Tumor Specimens

Excess tissue specimens from normal organs, 9 mesothelioma tumors (Table 2) , and 9 non-small cell lung carcinomas (NSCLC) were obtained freshly at the time of surgery and either immediately frozen in liquid nitrogen or frozen on dry ice after embedding in O.C.T. compound (Miles Scientific, Elkhart, IN) . Samples were stored at -70°C until further analysis. All diagnoses for the tumors were made by the pathologists at the University of Pennsylvania based on conventional histological and clinical criteria. Mesothelioma tumors were stained immunohistochemically and were characteristically negative for LeuMl and CEA. Results are reported in Table 2 below.

Table 2

Sample Age Sex Histoloqic Type

MT1 56 M Epithelial malignant mesothelioma (MM) M MTT22 6 699 F F Epithelial MM

MT3 59 F Mixed MM

MT4 51 M Spindle Cell MM

MT5 61 M Mixed MM

MT6 72 M Fibrosarcomatous MM M MTT77 7 700 M M Inflammatory MM

MT8 65 M Epithelial MM

MT9 — 0 Benign fibrous tumor

D . Immunoblot Analysis

To determine if the WTl protein was expressed in mesothelioma cell lines, immunoblotting experiments were performed, as follows, on nuclear extracts using the H2 anti-WTl MAb prepared as described in Example 3 above. Nuclear extracts were prepared from cell lines using standard techniques [F. M. Ausubel et al, In Current Protocols in Molecular Biology. John Wiley and Sons, New York (1991) ] . The nuclear pellet was collected by centrifugation at 4000 rp for 15 minutes at 4°C, resuspended in 5 times the pellet volume in electrophoresis sample buffer (62.5 mM Tris-HCl, 2% SDS, 10% glycerol, pH 6.8), and boiled for 5 minutes. Seventy-five μl of nuclear extract was applied on a 10% SDS-polyacrylamide gel under reducing conditions. The separated proteins were transferred to a nitrocellulose membrane which was developed as previously described [K. A. Knudsen et al, Exp. Cell. Res.. 152:218-226 (1985)] using anti-WTl as a primary antibody and an alkaline phosphatase-coupled anti-mouse as the secondary antibody.

The H2 MAb recognized a 52 KDa protein from the COS-1 cells transfected with pCMVhuWTlcDNA. No WTl expression was seen in non-transfected cells or in LLl, a lung cancer cell. However, in the ML17, ML13, ML16, and ML14 mesothelioma cell lines the antibody recognized two (52 and 55 KDa) proteins in varying amounts.

E. Immunolocalization Studies 1. Immunofluorescence

In order to determine the cellular location of the WTl protein and to confirm the im unoblotting experiments, immunofluorescence staining was performed on some of the mesothelioma cell lines, as follows.

Cell lines ML13 and ML16 which express elevated levels of WTl mRNA (determined using conventional RT-PCR techniques) were analyzed and LLl used as a negative control, since it expressed almost ho WTl mRNA. An isotyped matched monoclonal antibody against the endothelial cell specific molecule, PECAM-1 was used as a non-reactive control. Confluent monolayers of cells grown on glass coverslips coated with 1% gelatin were processed as previously described [S. M. Albelda et al, cited above]. Immunofluorescence studies were performed with a 1:250 dilution of anti-WTl ascites and a 1/200 fluorescein-conjugated anti-mouse antibody (Cappell Labs, Malvern, PA) . The coverslips were evaluated under epifluorescence. COS-1 cells grown on coverslips and transfected with pCMVhuWTlcDNA, were used as a positive control.

COS-1 cells transfected with pCMVhuWTlcDNA stained strongly with the monoclonal anti-WTl H2 with expression confined to the nucleus. In contrast, the untransfected COS-1 cells showed only baseline fluorescence. A similar nuclear staining pattern has been seen in COS-1 cells transfected with WTlcDNA and stained with a polyclonal anti-WTl antibody [J. F. Tet al, cited above]. Clear nuclear staining with the anti- WTl H2 MAb was also seen in the ML13 and the ML16 mesothelioma cell lines. In contrast, the lung cancer cell line (LLl) which did not express any WTl mRNA did not stain positively with the anti-WTl antibody. No appreciable staining was seen with the control antibody on any of the cell lines tested indicating the specificity of WTl MAb.

2. Immunohistochemistry

In addition to evaluating WTl protein expression in cell lines, the WTl protein expression was evaluated in tissues by immunohistochemical staining.

Frozen sections from 5 mesotheliomas and 5 NSCLC solid tumor specimens were stained with anti-WTl MAb and a control MAb.

For immunohistochemistry, thin sections (5 μm) were prepared from frozen tissues embedded in O.C.T. , fixed in acetone at -20°C for 5 minutes and stored at - 70°C. Prior to staining, the sections were blocked with 5% horse serum in PBS and washed twice in PBS at room temperature. The sections were incubated with a 1/1000 dilution of primary WTl monoclonal antibody diluted in PBS/4% bovine serum albumin (BSA) for 1 hour at room temperature. Sections were washed twice in PBS/4% BSA, and then incubated for 30 minutes with a 1/1000 diluted biotinylated IgG horse antibody to mouse. The streptavidin-biotin ABC peroxidase detection system

(Vector, Burlingame, CA) was applied, followed by a 2 minute incubation with 3-amino-9-ethylcarbazole (AEC) (Zymed, Sa Francisco, CA) as the substrate. The sections were mounted and evaluated microscopically. Strong, primarily nuclear, staining was noted in a subset of identifiable neoplastic cells (5- 10%) in all 5 mesothelioma tumors. Nuclear staining was not observed with a control MAb. Immunohistochemical staining of WT-1 was not observed, in any of the 5 non- small cell lung carcinomas examined as illustrated for LC4 and LC8.

F. Results

Immunohistochemical staining of both the mesothelioma tumors and the cell lines with the anti-WTl monoclonal antibody, H2, further revealed that WTl protein is expressed abundantly. As predicted for a transcription factor, the WTl protein localized to the nucleus in a proportion of cells in culture and in tumors. Although the staining of WTl protein has been observed in F9 embryonic carcinoma cells and in K562

cells [A. Telerman et al, Oncogene. . 8:2545-2548 (1992)], immunohistochemical localization of WTl in human tissues has not been previously reported. The general pattern of the expression WTl protein was somewhat heterogeneous in mesothelioma tumors, however, WTl was consistently expressed in at least some cells of all the tumors examined. Immunoblot analysis of nuclear extracts from mesothelioma cell lines revealed the presence of a 52 KDa and a 54 KDa sized WTl proteins. Whether the two proteins represent alternatively spliced WTl iso-forms

[D. A. Haber et al, Proc. Natl. Acad. Sci. USA. 88:9618- 9622 (1991) ] or a single form differently processed in the cancer cells is not known.

Numerous modifications and variations of the present invention are included in the above-identified specification and are expected to be obvious to one of skill in the art. Such modifications and alterations to the compositions and processes of the present invention are believed to be encompassed in the scope of the claims appended hereto.

SEQUENCE LISTING

(1) GENERAL INFORMATION:

(i) APPLICANT: The Wistar Institute of Anatomy and

Biology

(ii) TITLE OF INVENTION: WTl Monoclonal Antibodies and Methods of Use Therefor

(iii) NUMBER OF SEQUENCES: 7

(iv) CORRESPONDENCE ADDRESS:

(A) ADDRESSEE: Howson and Howson

(B) STREET: Spring House Corporate Cntr

PO Box 457

(C) CITY: Spring House

(D) STATE: Pennsylvania

(E) COUNTRY: USA

(F) ZIP: 19477

(v) COMPUTER READABLE FORM:

(A) MEDIUM TYPE: Floppy disk

(B) COMPUTER: IBM PC compatible

(C) OPERATING SYSTEM: PC-DOS/MS-DOS

(D) SOFTWARE: Patentln Release #1.0,

Version #1.25

(Vi) CURRENT APPLICATION DATA:

(A) APPLICATION NUMBER: WO

(B) FILING DATE:

(C) CLASSIFICATION:

(vii) PRIOR APPLICATION DATA:

(A) APPLICATION NUMBER: US 08/234,783

(B) FILING DATE: 28-APR-1994

(viii) ATTORNEY/AGENT INFORMATION:

(A) NAME: Bak, Mary E.

(B) REGISTRATION NUMBER: 31,215

(C) REFERENCE/DOCKET NUMBER: WST48PCT

(ix) TELECOMMUNICATION INFORMATION:

(A) TELEPHONE: 215-540-9200

(B) TELEFAX: 215-540-5818

(2) INFORMATION FOR SEQ ID NO:l:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 633 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: unknown

(ii) MOLECULE TYPE: cDNA

(ix) FEATURE:

(A) NAME/KEY: CDS

(B) LOCATION: 1..630

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:l:

ATG AGA GGA TCG CAT CAC CAT CAC CAT CAC TCC ATG GGT 39 Met Arg Gly Ser His His His His His His Ser Met Gly 1 5 10

TCC GAC GTT CGT GAC CTG AAC GCA CTG CTG CCG GCA GTT 78 Ser Asp Val Arg Asp Leu Asn Ala Leu Leu Pro Ala Val 15 20 25

CCG TCC CTG GGT GGT GGT GGT GGT TGC GCA CTG CCG GTT 117 Pro Ser Leu Gly Gly Gly Gly Gly Cys Ala Leu Pro Val 30 35

AGC GGT GCA GCA CAG TGG GCT CCG GTT CTG GAC TTC GCA 156 Ser Gly Ala Ala Gin Trp Ala Pro Val Leu Asp Phe Ala 40 45 50

CCG CCG GGT GCA TCC GCA TAC GGT TCC CTG GGT GGT CCG 196 Pro Pro Gly Ala Ser Ala Tyr Gly Ser Leu Gly Gly Pro 55 60 65

GCA CCG CCG CCG GCA CCG CCG CCG CCG CCG CCG CCG CCG 234 Ala Pro Pro Pro Ala Pro Pro Pro Pro Pro Pro Pro Pro

70 75

CCG CAC TCC TTC ATC AAA CAG GAA CCG AGC TGG GGT GGT 273 Pro His Ser Phe lie Lys Gin Glu Pro Ser Trp Gly Gly 80 85 90

GCA GAA CCG CAC GAA GAA CAG TGC CTG AGC GCA TTC ACC 312 Ala Glu Pro His Glu Glu Gin Cys Leu Ser Ala Phe Thr 95 100

GTT CAC TTC TCC GGC CAG TTC ACT GGC ACA GCC GGA GCC 351 Val His Phe Ser Gly Gin Phe Thr Gly Thr Ala Gly Ala 105 110 115

TGT CGC TAC GGG CCC TTC GGT CCT CCT CCG CCC AGC CAG 390 Cys Arg Tyr Gly Pro Phe Gly Pro Pro Pro Pro Ser Gin 120 125 130

GCG TCA TCC GGC CAG GCC AGG ATG TTT CCT AAC GCG CCC 429 Ala Ser Ser Gly Gin Ala Arg Met Phe Pro Asn Ala Pro

135 140

TAC CTG CCC AGC TGC CTC GAG AGC CAG CCC GCT ATT CGC 468 Tyr Leu Pro Ser Cys Leu Glu Ser Gin Pro Ala lie Arg 145 150 155

AAT CAG GGT TAC AGC ACG GTC ACC TTC GAC GGG ACG CCC 507 Asn Gin Gly Tyr Ser Thr Val Thr Phe Asp Gly Thr Pro 160 165

AGC TAC GGT CAC ACG CCC TCG CAC CAT GCG GCG CAG TTC 546 Ser Tyr Gly His Thr Pro Ser His His Ala Ala Gin Phe 170 175 180

CCC AAC CAC TCA TTC AAG CAT GAG GAT CCG GCT GCT AAC 585 Pro Asn His Ser Phe Lys His Glu Asp Pro Ala Ala Asn 185 190 195

AAA GCC CGA AAG GAA GCT GAG TTG GCT GCT GCC ACC GCT 624 Lys Ala Arg Lys Glu Ala Glu Leu Ala Ala Ala Thr Ala

200 205

GAG CAA TAA 633

Glu Gin 210

(2) INFORMATION FOR SEQ ID NO:2:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 210 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(Xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:

Met Arg Gly Ser His His His His His His Ser Met Gly Ser 1 5 10

Asp Val Arg Asp Leu Asn Ala Leu Leu Pro Ala Val Pro Ser 15 20 25

Leu Gly Gly Gly Gly Gly Cys Ala Leu Pro Val Ser Gly Ala 30 35 40

Ala Gin Trp Ala Pro Val Leu Asp Phe Ala Pro Pro Gly Ala 45 50 55

Ser Ala Tyr Gly Ser Leu Gly Gly Pro Ala Pro Pro Pro Ala 60 65 70

Pro Pro Pro Pro Pro Pro Pro Pro Pro His Ser Phe lie Lys

75 80

Gin Glu Pro Ser Trp Gly Gly Ala Glu Pro His Glu Glu Gin 85 90 95

Cys Leu Ser Ala Phe Thr Val His Phe Ser Gly Gin Phe Thr 100 105 110

Gly Thr Ala Gly Ala Cys Arg Tyr Gly Pro Phe Gly Pro Pro 115 120 125

Pro Pro Ser Gin Ala Ser Ser Gly Gin Ala Arg Met Phe Pro 130 135 140

Asn Ala Pro Tyr Leu Pro Ser Cys Leu Glu Ser Gin Pro Ala

145 150 lie Arg Asn Gin Gly Tyr Ser Thr Val Thr Phe Asp Gly Thr 155 160 165

Pro Ser Tyr Gly His Thr Pro Ser His His Ala Ala Gin Phe 170 175 180

Pro Asn His Ser Phe Lys His Glu Asp Pro Ala Ala Asn Lys 185 190 195

Ala Arg Lys Glu Ala Glu Leu Ala Ala Ala Thr Ala Glu Gin 200 205 210

(2) INFORMATION FOR SEQ ID NO:3:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 1680 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: unknown

(ii) MOLECULE TYPE: cDNA

(ix) FEATURE:

(A) NAME/KEY: CDS

(B) LOCATION: 381..1670

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:

GTTCAAGGCA GCGCCCACAC CCGGGGGCTC TGCGCAACCC GACCGCCTGT 50

CCGCTCCCCC ACTTCCCGCC CTCCCTCCCA CCTACTCATT CACCCACCCA 100

CCCACCCAGA GCCGGGACGG CAGCCCAGGC GCCCGGGCCC CGCCGTCTCC 150

TCGCCGCGAT CCTGGACTTC CTCTTGCTGC AGGACCCGGC TTCCACGTGT 200

GTCCCGGAGC CGGCGTCTCA GCACACGCTC CGCTCCGGGC CTGGGTGCCT 250

ACAGCAGCCA GAGCAGCAGG GAGTCCGGGA CCCGGGCGGC ATCTGGGCCA 300

AGTTAGGCGC CGCCGAGGCC AGCGCTGAAC GTCTCCAGGG CCGGAGGAGC 350

CGCGGGGCGT CCGGGTCTGA GCCTCAGCAA ATG GGC TCC GAC GTG 395

Met Gly Ser Asp Val 1 5

CGG GAC CTG AAC GCG CTG CTG CCC GCC GTC CCC TCC CTG 434 Arg Asp Leu Asn Ala Leu Leu Pro Ala Val Pro Ser Leu

10 15

GGT GGC GGC GGC GGC TGT GCC CTG CCT GTG AGC GGC GCG 473 Gly Gly Gly Gly Gly Cys Ala Leu Pro Val Ser Gly Ala 20 25 30

GCG CAG TGG GCG CCG GTG CTG GAC TTT GCG CCC CCG GGC 512 Ala Gin Trp Ala Pro Val Leu Asp Phe Ala Pro Pro Gly 35 40

GCT TCG GCT TAC GGG TCG TTG GGC GGC CCC GCG CCG CCA 551 Ala Ser Ala Tyr Gly Ser Leu Gly Gly Pro Ala Pro Pro 45 50 55

CCG GCT CCG CCG CCA CCC CCG CCG CCG CCG CCT CAC TCC 590 Pro Ala Pro Pro Pro Pro Pro Pro Pro Pro Pro His Ser 60 65 70

TTC ATC AAA CAG GAG CCG AGC TGG GGC GGC GCG GAG CCG 629 Phe lie Lys Gin Glu Pro Ser Trp Gly Gly Ala Glu Pro

75 80

CAC GAG GAG CAG TGC CTG AGC GCC TTC ACT GTC CAC TTT 668 His Glu Glu Gin Cys Leu Ser Ala Phe Thr Val His Phe 85 90 95

TCC GGC CAG TTC ACT GGC ACA GCC GGA GCC TGT CGC TAC 707 Ser Gly Gin Phe Thr Gly Thr Ala Gly Ala Cys Arg Tyr 100 105

GGG CCC TTC GGT CCT CCT CCG CCC AGC CAG GCG TCA TCC 746 Gly Pro Phe Gly Pro Pro Pro Pro Ser Gin Ala Ser Ser 110 115 120

GGC CAG GCC AGG ATG TTT CCT AAC GCG CCC TAC CTG CCC 785 Gly Gin Ala Arg Met Phe Pro Asn Ala Pro Tyr Leu Pro 125 130 135

AGC TGC CTC GAG AGC CAG CCC GCT ATT CGC AAT CAG GGT 824 Ser Cys Leu Glu Ser Gin Pro Ala lie Arg Asn Gin Gly

140 145

TAC AGC ACG GTC ACC TTC GAC GGG ACG CCC AGC TAC GGT 863 Tyr Ser Thr Val Thr Phe Asp Gly Thr Pro Ser Tyr Gly 150 155 160

CAC ACG CCC TCG CAC CAT GCG GCG CAG TTC CCC AAC CAC 902 His Thr Pro Ser His His Ala Ala Gin Phe Pro Asn His 165 170

TCA TTC AAG CAT GAG GAT CCC ATG GGC CAG CAG GGC TCG 941 Ser Phe Lys His Glu Asp Pro Met Gly Gin Gin Gly Ser 175 180 185

CTG GGT GAG CAG CAG TAC TCG GTG CCG CCC CCG GTC TAT 980 Leu Gly Glu Gin Gin Tyr Ser Val Pro Pro Pro Val Tyr 190 195 200

GGC TGC CAC ACC CCC ACC GAC AGC TGC ACC GGC AGC CAG 1019 Gly Cys His Thr Pro Thr Asp Ser Cys Thr Gly Ser Gin

205 210

GCT TTG CTG CTG AGG ACG CCC TAC AGC AGT GAC AAT TTA 1058 Ala Leu Leu Leu Arg Thr Pro Tyr Ser Ser Asp Asn Leu 215 220 225

TAC CAA ATG ACA TCC CAG CTT GAA TGC ATG ACC TGG AAT 1097 Tyr Gin Met Thr Ser Gin Leu Glu Cys Met Thr Trp Asn 230 235

CAG ATG AAC TTA GGA GCC ACC TTA AAG GGA CAC AGC ACA 1136 Gin Met Asn Leu Gly Ala Thr Leu Lys Gly His Ser Thr 240 245 250

GGG TAC GAG AGC GAT AAC CAC ACA ACG CCC ATC CTC TGC 1175 Gly Tyr Glu Ser Asp Asn His Thr Thr Pro lie Leu Cys 255 260 265

GGA GCC CAA TAC AGA ATA CAC ACG CAC GGT GTC TTC AGA 1214 Gly Ala Gin Tyr Arg lie His Thr His Gly Val Phe Arg

270 275

GGC ATT CAG GAT GTG CGA CGT GTG CCT GGA GTA GCC CCG 1253 Gly lie Gin Asp Val Arg Arg Val Pro Gly Val Ala Pro 280 285 290

ACT CTT GTA CGG TCG GCA TCT GAG ACC AGT GAG AAA CGC 1292 Thr Leu Val Arg Ser Ala Ser Glu Thr Ser Glu Lys Arg 295 300

CCC TTC ATG TGT GCT TAC CCA GGC TGC AAT AAG AGA TAT 1331 Pro Phe Met Cys Ala Tyr Pro Gly Cys Asn Lys Arg Tyr 305 310 315

TTT AAG CTG TCC CAC TTA CAG ATG CAC AGC AGG AAG CAC 1370 Phe Lys Leu Ser His Leu Gin Met His Ser Arg Lys His 320 325 330

ACT GGT GAG AAA CCA TAC CAG TGT GAC TTC AAG GAC TGT 1409 Thr Gly Glu Lys Pro Tyr Gin Cys Asp Phe Lys Asp Cys

335 340

GAA CGA AGG TTT TCT CGT TCA GAC CAG CTC AAA AGA CAC 1448 Glu Arg Arg Phe Ser Arg Ser Asp Gin Leu Lys Arg His 345 350 355

CAA AGG AGA CAT ACA GGT GTG AAA CCA TTC CAG TGT AAA 1487 Gin Arg Arg His Thr Gly Val Lys Pro Phe Gin Cys Lys 360 365

ACT TGT CAG CGA AAG TTC TCC CGG TCC GAC CAC CTG AAG 1526 Thr Cys Gin Arg Lys Phe Ser Arg Ser Asp His Leu Lys 370 375 380

ACC CAC ACC AGG ACT CAT ACA GGT GAA AAG CCC TTC AGC 1565 Thr His Thr Arg Thr His Thr Gly Glu Lys Pro Phe Ser 385 390 395

TGT CGG TGG CCA AGT TGT CAG AAA AAG TTT GCC CGG TCA 1604 Cys Arg Trp Pro Ser Cys Gin Lys Lys Phe Ala Arg Ser

400 405

GAT GAA TTA GTC CGC CAT CAC AAC ATG CAT CAG AGA AAC 1643 Asp Glu Leu Val Arg His His Asn Met His Gin Arg Asn 410 415 420

ATG ACC AAA CTC CAG CTG GCG CTT TGAGGGGTCT CCC 1680 Met Thr Lys Leu Gin Leu Ala Leu 425

(2) INFORMATION FOR SEQ ID NO:4:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 429 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:

Met Gly Ser Asp Val Arg Asp Leu Asn Ala Leu Leu Pro Ala 1 5 10

Val Pro Ser Leu Gly Gly Gly Gly Gly Cys Ala Leu Pro Val 15 20 25

Ser Gly Ala Ala Gin Trp Ala Pro Val Leu Asp Phe Ala Pro 30 35 40

Pro Gly Ala Ser Ala Tyr Gly Ser Leu Gly Gly Pro Ala Pro 45 50 55

Pro Pro Ala Pro Pro Pro Pro Pro Pro Pro Pro Pro His Ser 60 65 70

Phe lie Lys Gin Glu Pro Ser Trp Gly Gly Ala Glu Pro His

75 80

Glu Glu Gin Cys Leu Ser Ala Phe Thr Val His Phe Ser Gly 85 90 95

Gin Phe Thr Gly Thr Ala Gly Ala Cys Arg Tyr Gly Pro Phe 100 105 110

Gly Pro Pro Pro Pro Ser Gin Ala Ser Ser Gly Gin Ala Arg 115 120 125

Met Phe Pro Asn Ala Pro Tyr Leu Pro Ser Cys Leu Glu Ser 130 135 140

Gin Pro Ala lie Arg Asn Gin Gly Tyr Ser Thr Val Thr Phe

145 150

Asp Gly Thr Pro Ser Tyr Gly His Thr Pro Ser His His Ala 155 160 165

Ala Gin Phe Pro Asn His Ser Phe Lys His Glu Asp Pro Met 170 175 180

Gly Gin Gin Gly Ser Leu Gly Glu Gin Gin Tyr Ser Val Pro 185 190 195

Pro Pro Val Tyr Gly Cys His Thr Pro Thr Asp Ser Cys Thr 200 205 210

Gly Ser Gin Ala Leu Leu Leu Arg Thr Pro Tyr Ser Ser Asp

215 220

Asn Leu Tyr Gin Met Thr Ser Gin Leu Glu Cys Met Thr Trp 225 230 235

Asn Gin Met Asn Leu Gly Ala Thr Leu Lys Gly His Ser Thr 240 245 250

Gly Tyr Glu Ser Asp Asn His Thr Thr Pro lie Leu Cys Gly 255 260 265

Ala Gin Tyr Arg lie His Thr His Gly Val Phe Arg Gly lie 270 275 280

Gin Asp Val Arg Arg Val Pro Gly Val Ala Pro Thr Leu Val

285 290

Arg Ser Ala Ser Glu Thr Ser Glu Lys Arg Pro Phe Met Cys 295 300 305

Ala Tyr Pro Gly Cys Asn Lys Arg Tyr Phe Lys Leu Ser His 310 315 320

Leu Gin Met His Ser Arg Lys His Thr Gly Glu Lys Pro Tyr 325 330 335

Gin Cys Asp Phe Lys Asp Cys Glu Arg Arg Phe Ser Arg Ser 340 345 350

Asp Gin Leu Lys Arg His Gin Arg Arg His Thr Gly Val Lys

355 360

Pro Phe Gin Cys Lys Thr Cys Gin Arg Lys Phe Ser Arg Ser 365 370 375

Asp His Leu Lys Thr His Thr Arg Thr His Thr Gly Glu Lys 380 385 390

Pro Phe Ser Cys Arg Trp Pro Ser Cys Gin Lys Lys Phe Ala 395 400 405

Arg Ser Asp Glu Leu Val Arg His His Asn Met His Gin Arg 410 415 420

Asn Met Thr Lys Leu Gin Leu Ala Leu

425

(2) INFORMATION FOR SEQ ID NO:5:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 33 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: unknown

(ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5: CATGAGAGGA TCGCATCACC ATCACCATCA CTC 33

(2) INFORMATION FOR SEQ ID NO:6:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 33 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: unknown

(ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6: CATGGAGTGA TGGTGATGGT GATGCGATCC TCT 33

(2) INFORMATION FOR SEQ ID NO:7:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 17 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: unknown

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:

Val Ala Ala Gly Ser Ser Ser Ser Val Lys Trp Thr Glu Gly 1 5 10

Gin Ser Asn 15