AU596670B2 - Assays and antibodies for n-myc proteins - Google Patents

Description

AU-AI-74803/87 PCT WORLD INTELLECTUAL PROPERTY ORGANIZATION Interi au

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INTERNATIONAL APPLICATION PUBLISHED D f TH AT P ON TREATY (PCT) (51) International Patent Classification 4 (11) International Publication Number: WO 87/ 06940 CO7K 7/06, 7/08, 7/10 C07K 15/12, A61K 39/395 Al (43) International Publication Date: SGON 33/53, C12N 15/00 19 November 1987 (19.11.87) (21) International Application Number: PCT/US87/01046 (74) Agent: HESLIN, James, Townsend and Townsend, One Market Plaza, 2000 Steuart Tower, San (22) International Filing Date: 5 May 1987 (05.05.87) Francisco, CA 94105 (US).

(31) Priority Application Number: 860,276 (81) Designated States: AT (European patent), AU, BE (European patent), CH (European patent), DE (Euro- (32) Priority Date: 6 May 1986 (06.05.86) pean patent), FR (European patent), GB (European patent), IT (European patent), JP, LU (European pa- (33) Priority Country: US tent), NL (European patent), SE n4, patent).

SEcYION 34(4)(a) DIRECTION SEE FOLIO NAME DIRECTpT- UrifvQ A aQnc Ie Xnc O s 1i9 Gscc Scp :T e ll, Iqoo Qk klerrc, La.ne. do5 €2om ra S9J 24 DEC 1987 (72) Inventor: SLAMON, Dennis, 23122 Calvert Street, A Woodland Hills, CA 91367 AUSTRALIAN 1 DEC 1987 PATENT OFFICE (54) Title: ASSAYS AND ANTIBODIES FOR N-myc PROTEINS (57) Abstract Methods and compositions for identifying patients suffering from cancer, particularly neural and neuroendocrine cancers. It has been found that the protein expression product of the human N-myc photo-oncogene may be detected in certain biological specimens, particularly tissue specimens and sputum samples. By obtaining immunogenic N-myc polypeptides, either synthetically or by isolation from a natural source, antibodies specific for the N-myc protein are obtained.

Those antibodies may then be used in immunological techniques for detecting the presence of N-myc in the biological samples. In particular, the antibodies may be employed in immunohistochemical techniques to detect the N-myc protein in prepared tissue and sputum samples.

I:TI'1 is c!Qt~nt Cola:s tl ancndrnnnts made under Iion 49 and is corrct for pfrr.i ng.

WO 87106940 PCT/US87/01046 1 ASSAYS AND ANTIBODIES FOR N-myc PROTEINS BACKGROUND OF THE INVENTION 1. Field of the Invention Oncogenes are genes that, when activated or altered, may be involved in transformation of normal cells to a neoplastic phenotype. Numerous oncogenes have been identified in both humans and animals, and the transformation mechanisms associated with each vary widely. Frequently, the oncogene has a corresponding normal cellular gene referred to as a proto-oncogene, and modification of either the expression or the structure of the proto-oncogene may result in neoplastic transformation. For example, alteration of a single nucleotide in the ras proto-oncogene renders the host cell neoplastic, while transposition of the c-myc proto-oncogene results in increased expression and neoplastic transformation of the host. Another such example would be translocation of the c-abl proto-oncogene which results in a unique transcript and protein found in chronic myelogenous leukemia (CML).

N-myc is a human proto-oncogene which was originally identified because of its similarity to the viral c-myc oncogene. Gene amplification and/or increased expression of N-myc has been found in certain tumor cell lines as well as both primary and metastatic tumors. In particular, N-myc has been associated with neuroblastomas, retinoblastomas, and small-cell lung cancers (SCLC). Additionally, expression of N-myc has been found to complement mutant ras genes in tumorigenic conversion of rat fibroblasts. Thus, N-myc appears to be involved in the pathogenesis of certain human malignancies.

It would be desirable to isolate and characterize the N-myc gene product and to provide assay methods and reagent compositions useful for identifying and diagnosing tumors in which N-myc is expressed. In 7 2 particular, it would be desirable to obtain antigenic reagents capable of eliciting antibodies specific for the N-myL~ gene product and to use such antibodies in diagnosis and treatment of N-nVy related cancers.

2. Description of Pertinent References N-myc proto-oncogenes were first identified in human neuroblastoma cell lines which showed a 25 to 700-fold amplification of the gene. Schwab et al. (1983) Nature 305:245, and Kohl et al. (1983) Cell 35:359. The sequence of the N-myc in untreated primary neuroblastomas and retinoblastomas are reported in Brodeur et al. (1984) Science 224:1121; Seeger et al. (1985) New Engl. J. Med. 313:1111; and Lee et al. (1984) Nature 309:458. Amplification and increased transcription of N-myc have also been observed in human small-cell lung cancers. Nau et al. (1986) Proc. Natl. Acad. Sci. USA 83:1092-1096.

SUMMARY OF THE INVENTION The present invention provides methods and compositions for identifying and monitoring human cancers, particularly cancers which have neural or neuroendocrine properties such as neuroblastomas, retinoblastomas, and small-cell lung cancers. The methods rely on oo detection of N-myc protein in a biological specimen, usually a cell sample 20 such as a tissue sample or sputum sample. Presence of the N-myc protein in the biological specimen may be diagnostic and/or prognostic of the cancer.

Thus according to one embodiment of the invention there is provided an antigenic or haptenic polypeptide produced by expression in cultured cells of a fragment of the human N-myc gene, wherein the amino acid sequence of said polypeptide is selected from the gro,.p consisting of: AFGLGGLGGL TPNPVILQDC

MWSGFSAREK

LERAVSEKLQ HGRG; (II) ELAHPAAECV DPAVVFPFPV NKREPAPVPA APASAPAAGP AVASGAGIAA PAGAPGVAPP RPGGRQTSGG DHKALS; (III) ELAHPAAECV DPAVVFPFPV NKREPAPVPA APASAPAAGP AVASGAGIAA PAGAPGVAPP RPGGRQTSGG DHKALSTSGE DTLSDSDDED

DEEEDEEEEI;

'TLH/876c 1

A

(IV) GEDTLSDSDD

RSSSNTKAVT

(V GRAQSSELIL

SEDAPPQKKI

SPRNSDSEDS

FLTLRDHVP; and (VI) ELVKNEKAAK

LLLEKEKLQA

2A

EDDEEEDEEE

TFTITVRPKN

KRCLPI HQQH

KSEASPRPLK

ERRRNHNILE

VVI LKKATEY RQQQLLKKI E

EIDVVTVEKR

AALGP;

NYAAPSPYVE

SVI PPKAKSL RQRRND LRSS VHS LQA E EHQ

HARTC.

Polypeptides and antibodies thereto are utilized for detecting the N-p.M proteins, where the polypeptides are associated with immunogenic sites on the protein. The polypeptides may be natural or synthetic and S will include at least six contiguous amino acids of the natural protein, 0 0.

e0..

S*

usually including at least 9 contiguous amino acids, more usually C S S. C

C

CS

S

C CC TLH/5761U 'q ni i;; WO 87/06940 PCT/US87/01046 3 including at least 12 contiguous amino acids, representing detectable epitope(s) on the N-myc protein. Such polypeptides include the N-myc protein in substantially pure form as well as fragments thereof. Monoclonal or polyclonal antibodies against the polypeptides are prepared by conventional techniques, and the antibodies and polypeptides are utilized in a variety of conventional immunologic assay and histochemical staining techniques for detection of N-myc protein in the biological specimen.

The present invention is particularly useful for distinguishing among morphologically similar tumor types. For example, it is frequently difficult to distinguish various round cell tumors of childhood, such as neuroblastomas, neuroepitheliomas, Ewing sarcomas, rahbdomyosarcomas, and lymphomas. Since the treatment required depends greatly on the nature of the tumor, it is important to be able to distinguish the tumor type at an early stage. Use of the present invention will allow early identification of neuroblastomas and other tumors expressing N-myc. The present invention will also be useful for identifying the cellular origin of micrometastases in non-primary sites such as lymph nodes and bone marrow, and the amount of the N-myc protein expressed, measured by the intensity of staining, may correlate with the disease prognosis. The present invention also promises to provide early detection of small cell lung cancers, particularly by detection of cancer cells in sputum and/or lung lavage samples.

In a particular aspect of the present invention, novel DNA molecules are provided for expressing the N-myc polypeptides. The DNA molecules include at least a portion of the N-myc gene under the transcriptional control of a heterologous promoter, and usually also include an origin of replication. The N-myc gene may be natural or synthetic, and the DNA

I

WO 87/06940 PCT/US87/01046 4 molecule expressed in vitro to produce the N-myc polypeptides.

BRIEF DESCRIPTION OF THE DRAWINGS Fig. 1 represents the nucleotide sequence together with the deduced amino acid sequence for the N-myc 1 clone described in the Experimental section hereinafter.

Fig. 2 illustrates the P414/936 plasmid used in expressing various N-myc 1 fragments, as described in detail in the Experimental section hereinafter.

DESCRIPTION OF THE SPECIFIC EMBODIMENTS According to the present invention, methods for identifying cancers in human patients rely on detection of the expression product of the human N-myc proto-oncogene in a biological specimen, typically body fluids, tissue, or sputum samples. Conveniently, detection is accomplished by immunolcgical techniques, such as immunohistochemical staining ot a cell sample employing either monoclonal or polyclonal antibodies specific for the N-myc protein. Detection of the N-myc protein is particularly useful in distinguishing specific cancers, such as neuroblastomas, from morphologically similar cancers and for the early detection and differential diagnosis of cancers, such as small-cell lung cancers.

N-myc is a human proto-oncogene which has been associated with the pathogenesis of a number of human tumors, particularly neural and neuroendocrine associated tumors such as neuroblastomas, retinoblastomas, and small-cell lung cancers. As demonstrated by the work reported herein, the expression product of the N-myc gene is a doublet phosphoprotein including polypeptides of about 62 kD and 64 kD, respectively, where it is believed that the 62 kD species represents the gene product prior to phosphorylation. These molecular weights, of course, are only approximate and subject to experimental error WO 87/06940 PCT/US87/01046 in the measurement techniques reported in the Experimental section hereinafter. The protein has a half-life of about 30 to 50 minutes, and is located within the cell nucleus where 40 to 50% is associated with the nuclear matrix. As further demonstrated herein, neoplastic transformation of certain neural and neuroendocrine associated cells is characterized by detectable expression of the N-myc protein.

The polypeptides of the present invention will be either haptenic or antigenic, including at least 6 amino acids, usually at least 9 amino acids, and more usually 12 or more amino acids found contiguously within the natural N-myc protein. The contiguous amino acids may be located within any region of either of the doublet polypeptides and will correspond to at least one epitopic site which is characteristic of the protein. By characteristic, it is meant that the epitopic site will allow immunologic detection of the N-myc protein in a cell sample with reasonable assurance, in most cases allowing N-myc to be immunologically distinguished from other related proteins, such as c-myc. In other cases, however, the epitopic site(s) will be cross-reactive with other proteins, such as c-myc.

As reported in greater detail in the Experimental section hereinafter, six particular polypeptides within the carboxyl terminal 394 amino acids of the putative N-myc protein (as reported by Kohl et al.

(1986), supra.) have been identified which are capable of eliciting antibodies useful in the present invention. The sequences of these polypeptides (using the standard single letter designation) are as follows: AFGLGGLGGL TPNPVILQDC MWSGFSAREK LERAVSEKLQ HGRG; (II) ELAHPAAECV DPAVVFPFPV NKREPAPVPA APASAPAAGP AVASGAGIAA PAGAPGVAPP RPGGRQTSGG DHKALS; III II I- I I II r_ WO 87/06940 PCT/US87/01046 6 (III) ELAHPAAECV DPAVVFPFPV NKREPAPVPA APASAPAAGP AVASGAGIAA PAGAPGVAPP RPGGRQTSGG DHKALSTSGE DTLSDSDDED

DEEEDEEEEI;

(IV) GEDTLSDSDD EDDEEEDEEE EIDVVTVEKR RSSSNTKAVT TFTITVRPKN AALGP; GRAQSSELIL KRCLPIHQQH NYAAPSPYVE SEDAPPQKKI KSEASPRPLK SVIPPKAKSL SPRNSDSEDS ERRRNHNILE RQRRNDLRSS FLTLRDHVP; and (VI) ELVKNEKAAK VVILKKATEY VHSLQAEEHQ LLLEKEKLQA RQQQLLKKIE HARTC.

Of these six polypeptides, it has been found that II and III are capable of eliciting antibodies having very high specificity and affinity for the N-myc protein and which are free from reactivity with c-myc and other proteins. In contrast, antibodies raised against polypeptide V appear to cross-react with c-myc protein.

While antibodies raised against all six polypeptides react'with N-myc in immunoprecipitation assays with lysates of neuroblastoma cell lines, only II, III, and V elicit antibodies which histochemically stain the whole cells under the conditions tested.

The N-myc polypeptides may be natural, i.e., the entire N-myc protein or fragments thereof isolated from a natural source, or may be synthetic. The natural polypeptides may be isolated from natural sources, such as neuroblastoma and retinoblastoma cell lines known to produce the N-myc protein, by conventional techniques such as affinity chromatography. Conveniently, polyclonal or monoclonal antibodies obtained according to the present invention may be used to prepare a suitable affinity column by well known techniques. Such techniques are taught, for example, in Hudson and Hay, Practical Immunology, Blackwell Scientific Publications, Oxford, United Kingdom, 1980, Chapter 8.

I I i I ew Fi i i I WO 87/06940 PCT/US87/01046 7 Synthetic polypeptides which are immunologically cross-reactive with the natural N-myc protein may be produced by either of two general approaches. First, polypeptides having fewer than about 50 amino acids, more usually fewer than about amino acids, may be synthesized by the well-known Merrifield solid-phase synthesis method where amino acids are sequentially added to a growing chain (Merrifield (1963) J. Am. Chem. Soc. 85:2149-2156).

The amino acid sequences of such synthetic polypeptides may be based on the sequence of Fig. 1 described in the Experimental section hereinafter, or on the sequence for the entire N-myc gene reported in Kohl et al.

(1986) supra.

The second and preferred method for synthesizing the polypeptides of the present invention involves the expression in cultured cells of recombinant DNA molecules encoding a desired portion of the N-myc gene. The N-myc gene may itself be natural or synthetic with the natural gene obtainable from cDNA or genomic libraries using available probes, such as pNb-1 (Schwab et al. (1983) Nature 305:245).

Alternatively, probes may be synthesized based oh the DNA sequences reported in Fig. 1, as described in the Experimental section hereinafter. Suitable cDNA and genomic libraries may be obtained from human cell lines known to contain the N-myc gene, such as the LA-N-5 and IMR-32 human neuroblastoma cell lines. Alternatively, polynucleotides may be synthesized by well-known techniques. For example, short single-stranded DNA fragments may be prepared by the phosphoramidite method described by Beaucage and Carruthers (1981) Tett.

Letters 22:1859-1862. A double-stranded fragment may then be obtained either by synthesizing the complementary strand and annealing the strands together under appropriate conditions or by adding the complementary strand using DNA polymerase with an WO 87/06940 PCT/US87/01046 8 appropriate primer sequence. Particular DNA sequences may be based on Fig. 1 herein, or on the sequence reported by Kohl et al. (1986), supra.

The natural or synthetic DNA fragments coding for a desired N-myc fragment will be incorporated in DNA constructs capable of introduction to and expression in an in vitro cell culture. Usually, the DNA constructs will be suitable for replication in a unicellular host, such as yeast or bacteria, but may also be intended for introduction and integration within the genome of cultured mammalian or other eukaryotic cell lines. DNA constructs prepared for introduction into bacteria or yeast will include a replication system recognized by the host, the N-myc DNA fragment encoding the desired polypeptide product, transcriptional and translational initiation regulatory sequences joined to the 5'-end of the N-myc DNA sequence, and transcriptional and translational termination regulatory sequences joined to the 3'-end of the N-myc sequence. The transcriptional regulatory sequences will include a heterologous promoter which is recognized by the host. Conveniently, available expression vectors which include the replication system and transcriptional and translational regulatory sequences together with an insertion site for the N-myc DNA sequence may be employed.

To be useful in the detection methods of the present invention, the polypeptides are obtained in substantially pure form, that is, typically about w/w or more purity, substantially free of interfering proteins and contaminants. Preferably, the N-myc polypeptides are isolated or synthesized in a purity of at least about 80% w/w and, more preferably, in at least about 95% w/w purity. Using conventional protein purification techniques, homogeneous polypeptides of at least 99% w/w can be obtained. For example, the proteins may be purified by use of the antibodies WO 87/06940 PCT/US87/01046 9 described hereinafter using immunoadsorbent affinity chromatography. Such affinity chromatography is performed by first linking the antibodies to the solid support and then contacting the linked antibodies with the source of the N-myc proteins, lysates of cells which naturally produce N-myc or which produce N-myc as a -result of introduction of a recombinant N-myc DNA molecule.

Once a sufficient quantity of N-myc polypeptide has been obtained, polyclonal antibodies specific for the N-myc protein may be produced by in vitro or in vivo techniques. In vitro techniques involve in vitro exposure of lymphocytes to the antigenic polypeptides, while in vivo techniques require the injection of the polypeptides into a wide variety of vertebrates. Suitable vertebrates are non-human, including mice, rats, rabbits, sheep, goats, and the like. Polypeptides having more than about amino acids, particularly more than about 50 amino acids, may serve directly as immunogens. If the polypeptide is smaller than about 10 kD, particularly less than about 6 kD, it may be necessary to join the polypeptide to a larger molecule to elicit the desired immune response. The immunogens are then injected into the animal according to the predetermined schedule, and the animals are bled periodically with successive bleeds having improved titer and specificity. The injections may be made intramuscularly, S1 intraperitoneally, subcutaneously, or the like, and usually an adjuvant, such as incomplete Freund's adjuvant, will be employed.

If desired, monoclonal antibodies can be obtained by preparing immortalized cell lines capable of producing antibodies having the desired specificity.

Such immortalized cell lines may be produced in a variety of ways. Conveniently, a small vertebrate, such as a mouse, is hyperimmunized with the desired WO 87/06940 PCT/US87/01046 antigen by the method just described. The vertebrate is then killed, usually several days after the final immunization, the spleens removed, and the spleen cells immortalized. The manner of immortalization is not critical. Presently, the most common technique is fusion with a myeloma cell fusion partner, as first described by Kohler and Milstein (1976) Eur. J.

Immunol. 6:511-519. Other techniques include EBV transformation, transformation with bare DNA, e.g., oncogenes, retroviruses, etc., or any other method which provides for stable maintenance of the cell line and production of monoclonal antibodies.

When employing fusion with a fusion partner, the manner of fusion is not critical and various techniques may be employed. Conveniently, the spleen cells and myeloma cells are combined in the presence of a nonionic detergent, usually polyethylene glycol, and other additives such as Dulbecco's Modified Eagle's Medium, for a few minutes. At the end of the fusion, the nonionic detergent is rapidly removed by washing the cells. The fused cells are promptly dispensed in small culture wells (usually in a microtiter plate) at relatively low density, ranging from about 1-5x10 5 per well, in a selective medium chosen to support growth of the hybrid cells while being lethal to the myeloma cells. Usually, the myeloma cell line has been mutated to be sensitive to a lethal agent, typically being HAT sensitive.

After sufficient time, usually from one to two weeks, colonies of hybrids are observed and plates containing hybrid positive wells are identified. The plates and wells having only one colony per well are selected, and supernatants from these wells are tested for binding activity against the N-myc protein or the isolated antigen. Once positive hybridomas are identified, the cell line can be maintained as viable cultures and/or by lyophilization and frozen storage.

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WO 87/06940 PCT/US87/01046 11 Depending on the desired use for the antibodies, further screening of the hybridomas may be desirable. Hybridomas providing high titers are desirable. Furthermore, cytotoxic antibodies, e.g., IgG2a, IgG2b, IgG3 and IgM, may be selected for use in therapeutic treatment c, colorectal cancers. For use in immunodiagnostic assays, antibodies having very high specificity for the antigenic site are desirable.

Once the desired hybridomas have been selected, monoclonal antibodies may be isolated from the supernatants of the growing colonies. The yield of antibodies obtained, however, is usually low. The yield may be nehanced by various techniques, such as injection of the hybxidoma cell line into the peritoneal cavity of a vertebrate host which accept the cells. Monoclonal antibodies may then be harvested from the ascites fluid or the blood. Proteinaceous and other contaminants will usually be removed from the monoclonal antibodies prior to use by conventional technique, chromatography, gel filtration, precipitation, extraction, or the like.

By properly selecting polypeptides used as the immunogen, antibodies having high specificity and affinity for the N-myc protein can be obtained. The polypeptide selected should represent one or more epitopic sites which are unique to the N-myc protein and which can distinguish N-myc from closely related proteins such as c-myc. Such unique epitopes are found on polypeptides II and III shown above.

The polypeptides and antibodies of the present invention may be used with or without modification. Frequently, the polypeptides and antibodies will be labelled by joining, either covalently or non-covalently, a substance which provides for a detectable signal. A wide variety of labels and conjugation techniques are known and are reported extensively in both the scientific and patent j WO 87/06940 PCT/US87/01046 12 literature. Suitable labels include radionuclides, enzymes, substrates, cofactors, inhibitors, fluorescers, chemiluminescers, magnetic particles, and the like. Patents teaching the use of such labels include U.S. Patent Nos. 3,817,837; 3,850,752; 3,939,350; 3,996,345; 4,277,437; 4,275,149; and 4,366,241.

Antibodies and polypeptides prepared as described above can be used in various immunological techniques for detecting N-myc protein in biological specimens, particularly cell samples such as neurocytes, retina cells, and small lung cells (neuroendocrine-derived) and body fluid samples, including blood, plasma, serum, urine, and the like.

Depending on the nature of the sample, both liquid phase assays and solid-phase immunohistochemical staining techniques will find use. Conveniently, immunohistochemical staining techniques may be used with cell samples including tissue samples, sputum, and lung lavage samples, For example, a tissue sample may be fixed in formalin, B-5, or other standard histological preservative, dehydrated and embedded in paraffin as is routine in any hospital pathology laboratory. Sections may then be cut from the paraffinized tissue block and mounted on glass slides.

The N-myc protein, if present, may then be detected in the nucleus by exposure with labelled anti-N-myc antibody or exposure to unlabelled anti-N-myc antibody and a labelled secondary antibody. Sputum and lavage samples are typically prepared in a similar manner where the sample is first dehydrated by exposure to a dehydrating agent, typically a low molecular weight alcohol.

Liquid phase immunoassays or Western Blot analysis will also find use in the detection of the N-myc protein particularly in body fluids when the protein is shed into such fluids, blood or urine.

13 Solid tissue and sputum samples may also be assayed in liquid phase systems by lysing the cellular sample in order to release the intracellular protein. Once the protein is released, the sample will be placed in a suitable buffer, the sample buffer subjected to a suitable immunoassay.

Numerous competitive and non-competitive immunoassays are available and described in the scientific and patent literature, for example Harlow and Lane, Antibodies, A Laboratory Manual Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, 1988.

The antibodies of the present invention may also find use in cancer therapy and other medical applications. For example, anti-N-myc antibodies, preferably human antibodies, may be coupled to toxins, such as diptheria toxin and the ricin A chain, and administered to patients suffering from neural and neuroendocrine cancers. The use of antibody conjugated toxins in cancer therapy is described generally in U.S. Patent Nos. 4,093,607; 4,340,535; 4,379,145; and 4,450,154. Antibodies alone may also find use in treatment, particularly by blocking or interrupting the a.ctivity of the N-my protein which contributes to the neoplastic phenotype.

The following experiments are offered by way of illustration and not by way of limitation.

EXPERIMENTAL

Examples 1. Preparation and Expression of Partial cDNA Clone of N-myc.

To characterize the N-my. gene and its gene product, a cDNA library Swas constructed from the LA-N-5 human neuroblastoma cell line using the Okayama-Berg vector (Okayama and Berg (1982) Mol. Cell Biol. 2:101).

Approximately 80,000 clones were screened with pNb-l which is a plasmid S containing a human N-myc fragment isolated from a neuroblastoma cell line (Schwab et al. (1983), Sujra.). One positive clone, designated N-my. 1, S was obtained. Dideoxy sequencing of N-my. 1 was performed on independent 30 M13 clones containing either overlapping fragments or TLH/876c WO 87/06940 PCT/US87/01046 14 complementary DNA strands. The nucleotide sequence together with the deduced amino acid sequence is shown in Fig. 1. The amino acids in Fig. 1 are numbered corresponding to the complete sequencing as reported by Kohl et al. (1986) Supra.

The largest open reading frame in N-myc 1 is 1182 nucleotides long and would encode the carboxyl terminal 394 amino acids of the putative N-myc protein.

Comparison of the N-myc 1 sequence and that reported by Kohl et al. (1986) Supra. reveals only one notable difference between the large open reading frame from the N-myc 1 clone and the published sequence. The third base of the codon for amino acid 226 in the N-myc 1 clone is a cytosine, while the first base of the next codon is a guanine. The published sequence shows a reversal of these two residues. This difference results in a change in amino acid 227; alanine instead of proline.

A series of vectors for expressing portions of the putative N-myc encoded protein in E. coli were constructed by fusing the 5'-region of a bovine growth hormone gene (bGH) in frame, to six different regions of the N-myc 1 clone as follows: Fusion Gene Amino Acid Nos.* Symbol* bGH/N-myc I 96-111 bGH/N-myc II 178-253 bGH/N-myc III 178-277 bGH/N-myc IV 226-280 bGH/N-myc V 311-409 bGH/N-myc VI 410-464 See Fig. 1.

:i i WO 87/06940 PCT/US87/01046 The three sites in the bGH gene used for the fusion to N-myc specific sequences were SstI, PstI, and HindIII, generating fusion proteins containing 76, 92, and 114 residues of the amino terminus of bGH, respectively.

Expression vector p414/936 (isert- uaifA a modified temperature-sensitive runaway plasmid containing transcription termination and translation termination signals in all three reading frames adjacent to a polylinker used for introduction of genes linked to a tryptophan synthetase promoter (Fig. 2), was used for bGH/N-myc constructions I, III, IV, V and VI. The basic components of the p414/936 vector are: 1) a selectable drug marker, Amp 2) transcription termination signal, and 3) a poly linker containing translation termination signals in all three reading frames shown in boxes. All five fusion genes expressed using this vector were promoted by a tryptophan synthetase promoter depicted by the black box. The bGH/N-myc genes, including the promoter, were all cloned into the EcoRI site 5' to the promoter and at either the HpaI, BamHI or SstII sites (underlined) at the 3'-end of the genes. The specific 3'-cloning site is shown in brackets beside each construct. The shaded portion of each bar construct represents the bGH domain, while the open portion represents the N-myc domain. The number of amino acids representing either bGH or N-myc in each construct is shown in brackets within each bar construct. In addition, the letters opposite the bracketed numbers in the shaded portion refer to the restriction endonuclease sites in bGH where the N-myc gene segments were fused: H (HindIII), S (SstI), and P (Pstl). bGH/N-myc I was constructed by cloning the MluI/BamHI fragment (Fig. 1) of N-myc into M13mp20. The N-myc fragment was then recovered by cleavage with HindIII and SstII for cloning into the p 4 1 4 /93 6 expression vector with the appropriate bGH fragment (EcoRI/HindIII). bGH/N-myc III was generated TLH/5761U I I WO 87/06940 PCT/US87/01046 16 by cleaving N-myc 1 with TagI and blunt ending the site by filling in the appropriate nucleotides with Klenow.

N-myc 1 was then cut with SstI and the N-myc fragment ligated to the vector as shown above. bGH/N-myc IV was constructed by cloning the small N-myc MspI fragment into M13mpl0 (AccI) and recovering it by cleavage with PstI and BamHI. bGH/N-myc V was formed in the same manner as bGH/N-myc IV, using the largest MspI fragment of N-myc 1. Finally, bGH/N-myc VI was generated using an MspI/HindII fragment of N-myc 1 cloned into M13mp8 (AccI/HindII). The fragment was recovered using HindIII and HindII for cloning into the vector. The dotted portion of the bar construction for bGH/N-myc VI represents the 3'-untranslated sequence present in this construction.

Fusion gene bGH/N-myc II was expressed using vector pCFM414 previously described for obtaining expression of the human c-myb antigen. bGH/N-myc II was constructed by ligating an SstI/RsaI fragment of N-myc 1 in frame with the SstI site in the bGH gene and allowing the encoded product to terminate at a stop codon provided in the vector.

The above-described vectors were then expressed in E. coli as follows.

2. Preparation of Antisera in Rabbits The bGH/N-myc fusion proteins obtained as described above were used to produce polyclonal antisera in rabbits by the method described by Slamon Set al. (1985) Science 228:1427.

3. Characterization of the N-myc Protein Using the Polyclonal Antisera.

Antisera generated to the various N-myc protein tragments were tested in a liquid phase immunoprecipitation assay using human neuroblastoma cell lines (LA-N-5 and IMR-32) known to express N-myc transcripts, but not c-myc. The human promyelocytic cell line, HL-60, was used as a negative control, since h WO 870694 PCT/US87/01046 17 these cells express hign levels of c-myc transcripts, but not N-myc. In addition, two other-human cell lines, the HT 29 (colon carcinoma) and U251 (glioma), were used as negative controls since neither express N-myc transcripts. The criteria used to identify a protein as N-myc encoded were two-fold. First, the protein had to be immunoprecipitated from neuroblastoma cell lysates by antisera to at least two different N-myc-encoded tragments representing separate areas of the putative amino acid sequence, thus greatly reducing the possibility that the immunoprecipitation was due to chance sequence homology between the vector-expressed fragment and a cellular protein other than N-myc.

Second, the protein should not be found in other human tumor cell lines (including the neural derived glioma cells) where the N-myc transcript is not found.

Using these criteria, a protein appearing as a doublet of 62 kD and 64 kD was consistently immunoprecipitated from LA-N-5 human neuroblastoma cells by antibody directed to each of the six N-myc fragments generated in E. coli. Similar results were seen for the IMR-32 human neuroblastoma cell line.

Immunoprecipitation of this protein could be completely removed in all cases by competition with the appropriate N-myc fragment, indicating a specific antigenantibody reaction. Moreover, immunoprecipitation of the 62-64 kD doublet could not be blocked when non-homologous antibody and antigen combinations were used; attempts to block precipitation mediated by antisera to fragment II with fragment I antigen were unsuccessful, again indicating that the precipitation is due to a specific antigen-antibody reaction.

Finally, to ensure that the precipitation of the 62-64 kD protein was due to antibodies directed to the N-myc and not the bGH portion of the fusion protein, bGH alone was added to the reaction mix. In no instance r WO 87/06940 PCT/US87/01046 18 did the bGH inhibit the precipitation of the 62-64 kD protein.

The c-myc protein is similar in size (64-67 kD) and appearance (doublet) to the N-myc protein (Hann and Eisenman (1'984) Mol. Cell Biol. 4:2486). Evidence that this is not the c-myc protein, however, is provided by two sets of data. First, the protein identified using the anti-bGH/N-myc fusion antibodies is found in cells (LA-N-5) which lack detectable c-myc transcripts.

Second, five of the six antisera fail to identify any specific protein when used in immunoprecipitation assay with lysate from the HL-60 cell line which is rich in c-myc transcripts, indicating that they do not recognize the c-myc protein. The antisera directed against N-myc fragment V did recognize a 64-67 kD doublet in lysate from HL-.60 cells on long exposure (96 hours) of the immunoprecipitation radioautograph. Comparison of the deduced amino acid sequence from this fragment of N-myc and the analogous area in the c-myc protein shows significant sequence homology which would account for the ability of this antiserum to recognize the c-myc protein. None of the anti-bGH/N-myc antisera precipitated a 62-64 kD protein in the U251 or HT-29 control cell lines. Thus, the data demonstrate that the 62-64 kD protein is the N-myc-encoded protein.

Several aspects of the biochemistry and biology of the N-myc protein were investigated using the N-myc-specific antisera. To determine if the N-myc protein was phosphorylated, LA-N-5 cells were metabolically labelled in phosphate-free media with 32 3 2 P-orthophosphate (ICN Biochemicals, Inc., Irvine, CA) by the method of Osterman, in: Methods of Protein and Nucleic Acids Research, Springer-Verlag, New York, New York, 1984, pp. 144-150. The lysate was then subjected to immunoprecipitation using antisera to the bGH/N-myc II fusion protein and analyzed on SDS-PAGE gel. A band at 62-64 kD was found to be labelled with WO 87/06940 PCT/US87/01046 19 32 3 2 P-orthophosphate. Thus, like c-myc, the N-myc protein is a phosphoprotein.

The level of N-myc protein synthesis in neuroblastoma cells compared to other cellular proteins was estimated using an immunoprecipitation assay. In these experiments, a known amount of radioisotope-labelled whole cell lysate was subjected to immunoprecipitation using a combination of N-myc-specific antisera (anti-bGH/N-myc II and IV).

The resulting precipitated proteins were then subjected to SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel) analysis, and the doublet band at 62-64 kD was eluted from the gel, counted and compared to the total trichloroacetic acid-preciptable counts in the whole cell lysate as described by Slamon et al. (1985), supra. Approximately 0.076% of the total 35 S-methionine incorporation in these cells was associated with the 62-64 kD N-myc protein.

The kinetics of intracellular turnover of the N-myc protein were determined by pulse-chase labelling experiments. LA-N-5 human neuroblastoma cells were labelled with 3S-methionine for sixty minutes and chased with excess unlabelled methionine (Slamon et al.

(1985), supra.). The half-life of the p62-64 N my c doublet was between 30-50 minutes. Again, this feature of the N-myc protein is shared with c-myc, which has a half-life of 20-30 minutes.

To determine the subcellular localization of the N-myc protein, each of anti-N-myc antisera were used in immunocytochemical analyses of LA-N-5 cells.

Using an indirect immunoperoxidase staining procedure and dilutions of N-myc antisera of 1:2000 to 1:4000 by volume, a strong staining reaction was noted in the nuclei of the cells. All cells showed nuclear staining; however, variable intensity of the stain from cell to cell, indicating heterogeneity in N-myc protein content. While all six of the anti-N-myc antisera W m N I -0 i.

WO 87/06940 PCT/US87/01046 recognized the 62-64 kD protein in LA-N-5 cells by immunoprecipitation assay, under the cnditions tested' only three of these sera stained the neuroblastoma cells histochemically, anti-N-myc II, III and V. The fact that anti-N-myc sera I, IV and VI did not react immunocytochemically could be due to fixative-mediated alterations of the antigenic determinants to which the non-reacting antisera are directed. Conversely, these regions of the protein may be unavailable for recognition by the particular antisera, due to interactions of the antigenic regions with other cellular macromolecules or to the tertiary structure of N-myc within intact cells. To determine the specificity of the cytochemical reaction, HL-60 cells were used as a control cell line. Two of the three antisera (bGH N-myc II and III) did not stain HL-60 cells, indicating specificity for the N-myc protein. The antisera directed against N-myc fragment V stained both and HL-60 cells. As with the immunoprecipitation data, the ability of this antiserum to immunocytochemically recognize both the.N-myc and c-myc proteins is likely due to the high degree of homology between the two proteins in this region.

The nuclear localization of the 62-64 kD protein was confirmed by biochemical fractionation of cells into nuclear, cytoplasmic and membrane fractions as described by Slamon et al. (1985) supra.

The data placed the majority of the N-myc protein within the nucleus of neuroblastoma cells, and thus were cons;istent with those obtained by immunocytochemical analysis. Experiments to evaluate the subnuclear localization of the N-myc protein were done by obtaining intact nuclei from LA-N-5 cells and subjecting them to differential fractionation. The nuclei were fractionated into three components; nucleoplasm, chromatin and nuclear matrix as described by Boyle et al. (1985) Mol. Cell Biol. 5:3017. These fractions r- WO 87/06940 PCT/US87/01046 21 were then separately tested in immunoprecipitation assays using the N-myc specific anti-N-myc II antiserum. The majority of the N-myc protein was found associated with the nuclear matrix. Again, this is a feature which N-mc shares in common with c-myc.

Finally, to demonstrate the clinical diagnostic potential of the N-myc antisera, sections of primary human neuroblastoma tumors were tested using immunohistochemical techniques. In these experiments, only N-myc-specific antisera were used; i.e., anti-N-myc fragment II. As with cultured cell staining, the antiserum was used at dilutions of 1:2000 to 1:4000. Tissue from two separate primary tumors were examined; one from a patient with stage II disease, and one with stage IV disease. In both instances, there was intense and specific staining of malignant neuroblasts, with virtually no staining of the adjacent vascular or stromal tissue. The tumor from the patient with stage II disease was stroma rich, with nodular areas of undifferentiated cells. This tumor was shown to contain one copy of the N-myc oncogene by Southern Blot analysis (Seeger et al. (1985), Supra. The tumor from the patient with stage IV disease consisted of predominantly undifferentiated neuroblasts with surrounding vascular and stromal elements. It was shown to contain 200 copies of the N-myc oncogene (Id.) Although the foregoing invention has been described in some detail by way of illustration and Sexample for purposes of clarity of understanding, it will be obvious that certain changes and modifications may be practiced within the scope of the appended claims.

Claims (9)

1. An antigenic or haptenic r cultured cells of a fragment of the I acid sequence of said polypeptide is polypeptide produced by expression in human N-M~ gene, wherein the amino selected from the group consisting of: 00 *000 AFGLGGLGGL LERAVSEKLQ (ID) ELAHPAAECV APASAPAAGP RPGGRQTSGG (III)ELAHPAAECV APASAPAAGP RPGGRQTSGG DEEEDEEEEI; (IV) GEDTLSDSDD RSSSNTKAVT (V GRAQSSELIL SEDAPPQKKI SPRNSDSEDS FLTLRDHVP; and TPNPVILQDG HGRG; D PA VVFP FP V AVASGAGIAA DHKALS; D PA VVFP FP V AVASGAGIAA DHKALSTSGE N KR EPAP VPA PAGAPGVAPP N KR EPAP VPA PAGA PG VAP P DTLSDSDDED C S C 0 *0 C S C. C S. ewg MWSGFSAREK EDDEEEDEEE TFTITVRPKN KRCLP IHQQH KSEASPRPLK ERRRNHNILE EIDVVTVEKR AALGP; NYAAPSPYVE SVIPPKAKSL RQRRNDLRSS OC*@ *SSS *0*C CS S. S (VI) ELVKNEKAAK LLLEKEKLQA VVI LKKATEY RQQQLLKKI E VHS LQA EEHQ HARTC. *05 0 OS@* SOCS S C OS S C. C 0 0 0* 00 0 C SS *0so :1

2. Antibodies raised against the polypeptides of claim 1.

3. An immunological method for diagnosing neoplastic transformation comprising detecting human N-rny protein in a biological specimen, wherein tkhe N-Ruy protein is detected by exposing said specimen to anti-N-M~. antibodies. 1

4. A recombinant DNA molecule including a structural gene coding for a polypeptide according to claim 1, wherein said structural gene is 1-Ce-under transcriptional control of a heterologous promoter.

A recombinant DNA molecule as in claim 4, further including an TLH/5761U ;7 23 origin of replication recognized by a prokaryotic host.

6. A method for treating neoplastic diseases, said method comprising administering to a patient antibodies reactive with at least one epitope of the human N-myc protein, wherein said antibodies are raised against a fragment of the human N-myc gene comprising at least six amino acids.

7. A method as in claim 6, wherein the antibodies are conjugated to a toxin.

8. A method as in claim 6, wherein the antibodies are unconjugated.

9. An antigenic or haptenic polypeptide produced by expression in cultured cells of a fragment of the human N-myc gene, substantially as hereinbefore described with reference to the Examples. An immunological method for diagnosing neoplastic transformation, substantially as hereinbefore described with reference to S* the Examples. *see DATED this SEVENTH day of FEBRUARY 1990 The Regents of the Univeristy of California and Amgen, Inc. Patent Attorneys for the Applicant SPRUSON FERGUSON S. S S TLH/876c L A AGr q o ro pro ser tr l tr TCC GG CCCC GAGC I C IACG met leu leu glui GSAglu leu trp gly ATG GIG CIT GAG AAG GAG GTG TCC GGC ser pro ala glu g lu asp ala phe AGC CCG GCC GAG G AG GACG CG TIC N Iul 100 gly leu gly GGG GTG GGG 0 00 -4 0 C leo g gg ly leu thr CGGG CCICTCIGACC pro asn CCC AAq 110 pro vol CCG GIG ile leu gin asp cys5 met trp ser gly AIC GIG GAG GAG ICC AIC ICC AGGCCCG 120 130 p he ser ala org Il I ys leo gluo rg ala vol I C ICC GCC CCC GAG AAG GTG GAG CGC CC GIG 150 160 ro ala -li ala ala 5er ro la ly ara ~CG GA GCC G&C CC C CCAGG EI C*GC I G G I lu lys leuolgn his ly org ly GAG AAGCGG CAG CAC CCC CCC 6CCG Ssf 11 his gly gly ala ala gly ala gly GAC GGG CCCTCCC CI CCC C CAC 140 9 ro pro ihr a q ly ser thn ala- gln ser C CAACC GCCC T CCI CCACC GCCC CAGCICC ala gly ala ala leu GCC CG CC CC CCCG 180 190 po ala glu leu ala his pro ala al gI svol as p pro ala CC CC GAG GCC CC GAGC CC CCC GCCC GAG TUC GIG CAT CCC GCC S s IIBomilI 220 Vol Vol p he pro phe Pro Vol aso I ys rglol pro ala roVol pro ala ala pro ala ser oha pro ala ala 81Yp ro ala Vol ala GIG GCC;TICCC CC GIG AAG A G CC AG CCA GCGG CrC GG CGCA 6CC CC GCU AGT GCC CCG CC CCCG GC CCI GCG GICG6CC 0 la I I Ilile ala pr ly ala olC CC GGfa llCCal r ly a ~C po I Vol ala pro pro or8 proI Ia or aaOn thr ser a1v19lvasp his I s IC C O 6G T CC GCCA CCC GGCC CC M GIG CCC C CCCC C A MGG C CCAG ACC AGO GCC GAGGG ala leu ser thr ser gly glu asp fhr leu ser asp ser asp asp glu asp asp glo lu 9IN asp IN INlo lu glo ile asp Vol Vol CCC GIG ACT ACC ICC GGA GAG GAG ACC GIG AGC CAT ICA GA CAT GA A GAT CAT GAA GGAA CAT GAA GAG GAA CAA AICGAG GIG GIG RsaI spI Ta 91 FIG... i. (PART I OF 2) '4 K!' I- h lcol.. i IL.. IL.- L. *r iHe hr vml ora poa [s asn ala ala leU gly pro V thr vol ACT GIG 290 3003lOV glu lys org org ser ser ser aso Ihr lys ala vol thr thr ph. thr ile thr vol org pro iys 050 alo ala leu gly pro GAG AAG CCC CGI ICC TCC ICC AAC ACC AAG GOT GIG ACC ACA TIC ACC AIG ACT GIG CGT CCC AAG AAG GOA CCC GTG GI CCC IV rg~ GGT 8g ICC AGO GAG GIG i le leIJ ly ar cy le pr l his gin gi 050 s iyr ala ala pro ser pro tyr va G'GAGGGOTCAGTOOAGOGAGCT AT ONAAACGATGCCH CC TCGAG GAG CAG CAG AAG; TAT GCC CC CCC TCC COG TAC GIG 1AG 350 360 310 ser 91t; asp ala pro pro gin his lis Hle lys ser giu ala ser pro org pro ieu lys 5er vol i le pro pro Ivs ala I is ser ieu ACT GAG CAT GCA CCC CGA GAG AAG AAG ATA AAG AGG GAG C TCC CA CCI CCC GIG AAG ACT GIG ATO CCC CCA AAG GOT AAG AGO TIC 3er pro org osn ser asp ser glu asp ser Ilu org rg asn his oso Hi eu AGO CCC CA AAC TOT GAG TOG GAG GAC ACT GAG CCI CGC AGA AAG GAG AAC AICGI 390 or Al "r org osn asp icu ara ser CCC CCC AAC GAGOTCT CGG ICC 400 ser AGO phe iou thr leu org asp his vol II GIG ACG GOG ACG GAG GAG GIG pro glu leu Vol lys oso glu lys ala ala his vol vol He. leu lys lys ala thr qlu tyr vol CCC GAG TIC CIA AAG AAT GAG AAC COG CGCC AAC GIG CTC ATT TIC AAA AAG GC ACT GAG TAT GIG MSPI 440 450 460 his ser leu gin ala glu glu his gin leu leu leu glu lys glu lys leu gin ala org gin gin gin leu iou lys lys Hle glu his GAGC GIO GT AG CC GAG GAG GAG GAG OTT TTGCTGG GAA AAC GAA AAA TIGCGAG CCA ACA GAG GAG GAG TIC CIA AAC AAA ATT CAA GAG ala org thr c y AM GOT CCC ACT TCC TAG ACCCTCTCAAAACTGGAGAGTCACTGCACTTTCACATTTATTTTTIUTTAAACAAACATTGTGTTGACAI TAAGAATCTIGGCTIACTTTGAA HlindlE 1 (PART 2 OF 2) I I- WO 87/06940 WO 87~6940PCT/US87/0 1046 SstI p,414/936 toop I i I I I TAAATA EcoRI HpaI HindM BarnHI SstlI 1I H (114) 4) [sstfl] m (00 [HpaI] IV 755)7 am H I V P 101 01-N 9 2 MIN 100) [BamHI] FIG....26 SUBSTITUTE SHEET i INTERNATIONAL SEARCH REPORT International Application tCT/US 87/0104 6 L 1. CLASSIFICATION OF SUBJECT MATTER (If several classification symbols apply, Indicate all) According to International Patent Classfication (IPC) or to both National Clasficatlon and IPC IPC 4 C07K 7/0 7/08,7/10,15/12;A61K39/395;GOIN 33/53;C12N 15/00. U.S. I n/7A 7 '127 1928-_ q.9 0 387 402. 410 80 8 424/85 435/7.172 3 II. FIELDS SEARCHED Minimum Documentation Searched 4 Classification System I Clausification Symbols U.S. 30/324,326,327,328,329,350,387,402,410,808;424/85; p35/7, 172.3. Documentation Searched other than Minimum Documentation to the Extent that such Documents are Included In the Flelds Searched 6 CHEMICAL ABSTRACT AND BIOLOGICAL ABSTRACT ON LINE COMPUTER SEARC III. DOCUMENTS CONSIDERED TO BE RELEVANT ,4 Category Citation of Document, I( with indication, where appropriate, of the relevant passages I Relevant to Claim No. is ROC.,NATL. ACAD. SCI. USA. VOLUME 83 ISSUED 1-3,7, ARCH 1986, (STANTON ET AL), "NUCLEOTIDE 12-14,16 EQUENCE OF THE HUMAN N-MYC GENE", PAGES

1772-1776, SEE PAGE 1773, 1774, 1775 IN ARTICULAR. ,P SCIENCE (WASHINGTON, D. C. USA), VOLUME 232 1-19 ISSUED 09 MAY 1986, (SLAMON ET AL.), "IDENTIFICATION AND CHARACTERIZATION OF THE PROTEIN ENCODED BY THE HUMAN N-MYC ONCOGENE", PAGES 768-772. SEE PAGES 768, 769, 770, 771, 772. ELL VOLUME 35, ISSUED 1983, (KOHL ET AL), 10-11 "TRANSPOSITION AND AMPLIFICATION OF ONCOGENE -RELATED SEQUENCES IN HUMAN NEUROBLASTOMAS", PAGES 359-367. SEE PAGE 359 IN PARTICULAR. NATURE, (LONDON, ENGLAND), VOLUME 309, 10-11 ISSUED MAY 1984, (LEE ET AL), "EXPRESSION AND AMPLIFICATION OF THE N-MYC GENE IN PRIMARY RETINOBLASTOMA", PAGES 458-460. SEE PAGE 458 IN PARTICULAR. Special categories of cited documents: 'I later document published after the international filing date document defning the general state of the art which is not or priority date and not in conflict with the application but considered to he of particular relevance ncited to understand the principle or theory underlying the considered to be of particular relevance invention earlier document but published an or after the nternational document of particular relevance: the claimed Invention filing date cannot be considered novel or-cannot be considered to document which may throw doubts on priority claim(s) or involve an inventive step which s cited to establish the publication date of another document of particular relevance: the claimed invention citation or other special reason (as pecified) cannot be considered to involve an inventive step when the document referring to an oral disclosure, use, exhibition or document is combined with one or more other such docu- other means ments, such combination being obvious to a person skilled document published prior to the international filing date but in the art. later than the priority date claimed document member of the same patent family IV. CERTIFICATION Date of the Actual Completion of the International Search s Date of Mailing of this International Search Report I JULY 1987 0 4 AUG 1987 International Searching Authority t Signature of Authorized Officer to FIISA/Iq210 (ond het) (MTTTAy 1986___ Forri PCT/ISA/210 (second sheet) (May 19861 L, International Application No. PCT/US87/01046 III. DOCUMENTS CONSIDERED TO BE RELEVANT (CONTINUED FROM THE SECOND SHEET) Category* Citation of Document, ti with Indication, where appropriate, of the relevant passages 7 Relevant to Claim No X NATURE, (LONDON, ENGLAND), VOLUME 305, 10-11 ISSUED SEPTEMBER 1983, (SCHWAB ET AL), V/ "AMPLIFIED DNA WITH LIMITED HOMOLOGY TO MYC CELLULAR ONCOGENE IS SHARED BY HUMAN NEUROBLASTOMA CELL LINES AND A NEURO- BLASTOMA TUMOUR, PAGES 245-248. SEE PAGES 245-246 IN PARTICULAR. A NATURE, (LONDON, ENGLAND), VOLUME 319, ISSUED 1-19 JANUARY 1986, (KOHL ET AL), "HUMAN N-MYC IS CLOSELY RELATED IN ORGANIZATION AND NUCLEO- TIDE SEQUENCE TO C-MYC", PAGES 73-77. X SCIENCEL (WASHINGTON, USA), VOLUME 10-11 224, ISSUED 1984, (MARX), "THE N-MYC ONCOGENE IN NEURAL TUMORS", PAGE 1088 X PROC., NATL. ACAD. SCI. USA, VOLUME 83, 10-11 ISSUED 1986, (NAU ET AL), "HUMAN SMALL-CELL LUNG CANCERS SHOW AMPLIFICATION AND EX- PRESSION OF THE N-MYC GENE", PAGES 1092-1096. SEE PAGE 1092 IN PARTICULAR. X NEW ENGLAND JOURNAL OF MEDICINE, VOLUME 313, 10-11 ISSUED OCTOBER 1985, (SEEGER ET AL), "ASSOCIATION OF MULTIPLE COPIES OF THE N-MYC ONCOGENE WITH RAPID PROGRESSION OF NEURO- BLASTOMAS", PAGES 1111-1116. SEE PAGE 1111 IN PARTICULAR. X SCIENCEL (WASHINGTON, USA), VOLUME 10-11. 224, ISSUED 1984, (BRODEUR ET AL), "AMPLIFI- CATION OF N-MYC IN UNTREATED HUMAN NEURO- BLASTOMAS CORRELATES WITH ADVANCED DISEASE STAGE", PAGES 1121-1124. SEE PAGE 1121 IN PARTICULAR. X,P THE EMBO JOURNALCOXFORD, ENGLAND), VOLUME 5, 1-3, 7 ISSUED JUNE 1986, (TAYA ET AL), "NULEOTIDE 12-14,16 SEQUENCE OF THE CODING REGION OF THE MOUSE N-MYC GENE", PAGES 1215-1219, SEE FIG. 3 IN PARTICULAR. Form PCT/ISA/210 (extra sheet) (May 1986) P- C International Application No. PCT/US87/01046 FURTHER INFORMATION CONTINUED FROM THE SECOND SHEET V.Q OBSERVATIONS WHERE CERTAIN CLAIMS WERE FOUND UNSEARCHABLE o1 This international search report has not been established in respect of certain claims under Article 17(2) for the following reasons: 1.r Claim numbers because they relate to subject matter 12 not required to be searched by this Authority, namely: Claim because they relate to parts of the international application that do not comply with the prescribed require- ments to such an extent that no meaningful international sparch can be carried out 13, specifically: VI.X] OBSERVATIONS WHERE UNITY OF INVENTION IS LACKING II This International Searching Authority found multiple inventions In this international application as follows: I. Claims 1-4, 7 and 14-16 drawn to a polypeptide. II. Claims 5-6 and 17-19 drawn to an antibody and a method for treating disease. III. Claims 8-11 drawn to a method for diagnosing neoplastic transformation. IV. Claims 12-13 drawn to a recombinant DNA. As all required additional search fees were timely paid by the applicant, this international search report covers all searchable claims of the International application, Telephone ,r Telephone practice As only some of the required additional search fees weretlmel paid by the applicant, this international search report covers only those claims of the international application for which fees were paid, speciically claims: 3.Q No required additional search fees were timely paid by the applicant. Consequently, this international search report is restricted to the invention first mentioned in the claims; it is covered by claim numbers: 4. As all searchable claims could be searched without effort justifying an additional fee, the International Searching Authority did not invite payment of any additional fee. Remark on Protest The additional search fees were accompanied by applicant's protest. No protest accompanied the payment of additional search fees. Form PCT/ISA/210 (supplemental sheet (May 1986) -t