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Dna segment encoding a gene for a receptor related to the epidermal growth factor receptor

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CA2069900C
CA2069900C CA 2069900 CA2069900A CA2069900C CA 2069900 C CA2069900 C CA 2069900C CA 2069900 CA2069900 CA 2069900 CA 2069900 A CA2069900 A CA 2069900A CA 2069900 C CA2069900 C CA 2069900C
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erbb
dna
sequence
human
acid
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CA2069900A1 (en )
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Matthias H. Kraus
Stuart A. Aaronson
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US Department of Health and Human Services (HHS)
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    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K16/00Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/32Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against translation products of oncogenes
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL, OR TOILET PURPOSES
    • A61K47/00Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6835Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
    • A61K47/6843Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a material from animals or humans
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K14/00Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/82Translation products from oncogenes
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00Investigating or analysing materials by specific methods not covered by the preceding groups
    • G01N33/48Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53Immunoassay; Biospecific binding assay
    • G01N33/574Immunoassay; Biospecific binding assay for cancer
    • G01N33/5748Immunoassay; Biospecific binding assay for cancer involving oncogenic proteins
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00Investigating or analysing materials by specific methods not covered by the preceding groups
    • G01N33/48Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53Immunoassay; Biospecific binding assay
    • G01N33/574Immunoassay; Biospecific binding assay for cancer
    • G01N33/57484Immunoassay; Biospecific binding assay for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL, OR TOILET PURPOSES
    • A61K38/00Medicinal preparations containing peptides
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K2317/00Immunoglobulins specific features
    • C07K2317/30Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/34Identification of a linear epitope shorter than 20 amino acid residues or of a conformational epitope defined by amino acid residues
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00Screening for compounds of potential therapeutic value
    • G01N2500/02Screening involving studying the effect of compounds C on the interaction between interacting molecules A and B (e.g. A = enzyme and B = substrate for A, or A = receptor and B = ligand for the receptor)
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00Screening for compounds of potential therapeutic value
    • G01N2500/04Screening involving studying the effect of compounds C directly on molecule A (e.g. C are potential ligands for a receptor A, or potential substrates for an enzyme A)

Abstract

A DNA fragment distinct from the epidermal growth factor receptor (EGF-R) and erbB-2 genes was detected by reduced stringency hybridization of v-erbB to normal genomic human DNA.
Characterization of the cloned DNA fragment mapped the region of v-erbB homology to three exons with closest homology of 64 % and 67 % to a contiguous region within the tyrosine kinase domains of the EGF-R and erbB-2 proteins, respectively, cDNA cloning revealed a predicted 148 kd transmembrane polypeptide with structural features identifying it as a member of the erbB
family, prompting designation of the new gene as erbB-3.
It was mapped to human chromosome 12q11-13 and was shown to be expressed as 6.2 kb transcript in a variety of normal tissues of epithelial origin. Markedly elevated erbB-3 mRNA levels were demonstrated in certain human mammary tumor cell lines.
These findings indicate that increased erbB-3 expression, as in the case of EGF-R and erbB-2, plays a role in some human malignancies.

Description

--~?~O 91 /08214 - 1 - ~ ~ ~~ ~ ~ ~Iv PCT/US90/07025 DNA SEGMENT ENCODING A GENE FOR A RECEPTOR RELATED TO
THE EPIDERMAL GROWTH FACTOR RECEPTOR
FIELD OF THE INVENTION
The present invention relates to genes which encode novel proteins related to a family of receptor proteins typified by two related membrane scanning tyro sine kinases: the Epidermal Growth Factor receptor (EGF
R), which is encoded by the erbB gene, the normal human counterpart of an oncogene (v-erbB) that was first recog nized in the proviral DNA of avian erythroblastosis virus;
and the receptor encoded by the related gene erbB-2. In particular, the present invention relates to a DNA segment encoding the coding sequence, or a unique portion thereof, for a third member of this receptor gene family, herein designated erbB-3.
BACKGROUND OF THE INVENTION
Proto-oncogenes encoding growth factor receptors constitute several distinct families with close overall structural homology. The highest degree of homology is observed in their catalytic domains, essential for the intrinsic tyrosine kinase activity of these proteins.
Examples of such receptor families include: the EGF-R and the related product of the erbB-2 oncogene; the Colony Stimulating Factor 1 receptor (CSF-1-R) and the related Platelet-Derived Growth Factor receptor (PDGF-R); the insulin receptor (IF-R) and the related Insulin-like Growth factor 1 receptor (IGF-1-R); and the receptors encoded by the related oncogenes eph and elk.
It is well established that growth factor recep tors in several of these families play critical roles in regulation of normal growth and development. Recent studies in Drosophila have emphasized how critical and multifunctional are developmental processes mediated by ligand-receptor interactions. An increasing number of Drosophila mutants with often varying phenotypes have now been identified as being due to lesions in genes encoding such proteins. The genetic locus of the Drosophila EGF-R
homologue, designated DER, has recently been identified as W0 91 /08214 ~ ~ ~ . ' .
PCT/US90/07P=:

being allelic to the zygotic embryonic lethal faint little ball exhibiting a complex phenotype with deterioration of multiple tissue components of ectodermal origin. Fur-thermore, other mutants appear to lack DER function either in the egg or the surrounding maternal tissue. Thus, the DER receptor may play an important role in the ligand-receptor interaction between egg and follicle cells necessary for determination of correct shape of eggshell and embryo. It is not yet known whether DER represents the sole of the Drosophila counterpart of both known mammalian erbB-related genes.
Some of these receptor molecules have been impli-cated in the neoplastic process as well. In g~articular, both the erbB and erbB-2 genes have been shown to be activated as oncogenes by mechanisms involving over-expression or mutations that constitutively activate the catalytic activity of their encoded receptor proteins (Bargmann, C. I., Hung, M. C. & Weinberg, R. A., 1986, Cell 45:649-657; Di Fiore, P. P., Pierce, J. H., Rraus, M.
H., Segatto, O., Ring, C. R. & Aaronson, S. A., 1987, Science 237:178-182; Di Fiore, P. P., Pierce, J. H., Fleming, T. P., Hazan, R., Ullrich, A., King, C. R., Schlessinger, J. & Aaronson, S. A., 1987, Cell 51:1063-1070; Velu, T. J., Beguinot, L., Vass, W. C., Willingham, M. C., Merlino, G. T., Pastan, I. & Lowy, D. R., 1987, Science 238:1408-1410). Both erbB and erbB-2 have been casually implicated in human malignancy. erbB gene amplification or overexpression, or a combination of both, has been demonstrated in squamous cell carcinomas and glioblastomas (Libermann, T. A., Nusbaum, H. R., Razon, N., Kris, R., Lax, I., Soreq, H., Whittle, N., Waterfield, M. D., Ullrich, A. & Schlessinger, J., 1985, Nature 313:144-147). erbB-2 amplification and overexpression have been observed in human breast and ovarian carcinomas (King, C. R., Kraus, M. H. & Aaronson, S. A., 1985, Science 229:974-976; Slamon, D. J., Godolphin, W., Jones, L. A. , Holt, J. A. , Wong, S. G. , Keith, D. E. , Levin, W.
J., Stuart, S. G., Udove, J., Ullrich, A. & Press, M. F., ~'"'' 91/08214 G ~ 6 9 9 0 0 PCT/US90/07025 -~ 3 -1989, Science 244:707-712), and erbB-2 overexpression has been reported to be an important prognostic indicator of particularly aggressive tumors (Slamon, D. J., et al., 1989, supra). Yet, not all such tumors have been found to overexpress erbB-2, and many human tumors have not yet been associated with any known oncogene. Thus, there has been a continuing need to ;search for additional oncogenes which would provide knowledge and methods for diagnosis and, ultimately, for rational molecular therapy of human cancers.
BRIEF DES RIPT'ION OF THE DRAWINGS
Figures 1A and 1B show detection of v-erb8-related DNA fragments in DNAs from normal human thymus (lane 1), human maitnnary tumor lines :MDA-MB468 ( lane 2 ) , and SK-BR-3 (lane 3). Hybridization was conducted at reduced (Fig.
2A) or intermediate (Fig. 2B) stringency conditions. The arrow denotes a novel 9 k:ilobase pair ( kbp ) erbB-related restriction fragment distinct from those of the EGF-R gene (erbB) and erbB-2.
Figure 2 shows genomic and cDNA cloning of erbB-3.
The region of v-erbB homology within the genomic 9 kbp Sacl insert of ~E3-1 was subcloned into the plasmid pUC
(pE3-1) and subjected to nucleotide sequence analysis.
The three predicted exons are depicted as solid boxes.
erbB-3 cDNA clones were isolated from oligo dT-primed libraries of mRNAs from normal human placenta (shaded bars) and the breast tumor cell line MCF-7 (open bar).
The entire nucleotide sequence was determined for both strands on erbB-3 complementary DNA from normal human placenta and upstream of the 5' XhoI site,on pE3-16. The coding sequence is shown as a solid bar and splice junc-tions of the three characaerized genomic exons are indi-cated by vertical white :Lines. Solid lines in the cDNA
map represent untranslated sequences. Restriction sites:
A=AccI, Av=Aval, B=HamHI, Bg=9gIII, E=EcoRI, H=HindIII, K=KpnI, M=MstII, P=PstI, .S=Sacl, Sm=Smal, Sp=SpeI.
Figure 3 shows the nucleotide sequence of the 2 0 6i 9 9 0 0 P~~US90/p7025 region of v-erbB homology in the human erbB-3 gene derived from human genomic DNA clone E3-1, in the 1.5 kbp region from the EcoRI to the Pstl sites. This region contains three open reading frames bordered by splice junction consensus sequences (underlined). The predicted amino acid sequences of the three exons are shown in three letter code above the relevant DNA sequences.
Figure 4 shows the: nucleotide sequence of the cDNA
encoding the erbB-3 polypeptide and the predicted amino 1o acid sequence of that polypeptide.
Figure 5 shows comparison of the predicted amino acid sequence of the erb8-3 polypeptide with other recep-tor-like tyrosine kinases. The amino acid sequence is shown in single letter code and is numbered on the left.
The putative extracellular domain (light shading) extends between the predicted signal sequence (solid box) at the amino-terminus and a s~!ngle hydrophobic transmembrane region (solid box) within the polypeptide. The two cysteine clusters (Cys) in the extracellular domain and the predicted tyrosine kinase domain (TR) within the cytoplasmic portion of the polypeptides are outlined by dark shading. The putati~re ATP-binding site at the amino terminus of the TR domain is circled. Potential auto phosphorylation sites within the carboxyl-terminal domain (COON) are indicated by asterisks. Potential N-linked glycosylation sites (~---~) are marked above the amino acid sequence. The percentage of amino acid homology of erbB-3 in individual domains with erbB-2, EGF-R, met, eph, insulin receptor (IR), and fms is listed below. Less than 16% identity is denoted by (-).
Figure 6 shows the assignment of the genomic locus of erbB-3 was assigned to human chromosomal locus 12q13.
A total of 142 grains were localized on the 400-band ideogram. As depicted in the diagram, specific labeling of chromosome 12 was observed, where 38 out of 51 grains were localized to band q13.
Figures 7A and 7B show the elevated erbB-3 transcript levels in human mammary tumor cell lines. A
Northern blot containing 10 ~g total cellular RNA from E """'~ 91/08214 _ 5 _ PCT/US90/07025 AH589 mammary epithelial cells (lane 1), as well as mammary tumor cell lines 7MDA-MB415 (lane 2) and MDA-MB453 (lane 3) was hybridized with an erbB-3 cDNA probe (Fig.
7A). Following signal decay the same blot was rehybrid-ized with a human ~-actin cDNA probe (Gunning, P., Ponte, P., Okayama, H., Engel, ,;f., Blau, H. & Kedes, L., 1983, Mol. Cell Biol. 3:787-795).
Figures 8A and 8B show the expression of a human erbB-3 polypeptide in cells transformed by a cDNA segment as detected by an erbB-3--specific antipeptide antiserum.
Cellular lysates (100 ~g of each, sample) were electropho-resed and transferred to nitrocellulose membranes for analysis by Western blotting. Figure 8A shows the detec-tion of erbB-3 polypeptids: with the antiserum. Figure 8B
shows the preincubation of the antiserum with homologous peptide. Antibody blocking indicates binding specificity.
Lane 1: Selected cultures of NIH3T3 cells transfected with 1 ~g LTRerbB-3 expression vector. Lane 2: control NIH3T3 cells.
SUMMARY OF THE INVENTION
It is an object of the present invention to provide a DNA segment encoding a receptor protein related to the erbB proto-oncogene family which previously has not been known or even suspected to exist. Further, it is an object of the present invention to develop assays for expression of the RNA and protein products of such genes to enable determining whet:hc~r abnormal expression of such genes is involved in human cancers.
In pursuit of the above objects, the present inventors have discovered a human genomic DNA fragment that is produced by cleavage with the SacI restriction enzyme, has a size of about 9 kbp, and is detectable by nucleic acid hybridization with a probe derived from the v-erbB gene only under reduced stringency hybridization conditions. Thus, this DNA fragment is distinct from those known to encode the epider~aal growth factor receptor (EGF-R) (i.e., the erbB gene) and from the related erbH-2 gene. Characterization of this DNA fragment after partial 20Ei9900 . d0 91 /08214 _ g - PCT/ US90/070Z5 purification and molecular cloning showed that the region of v-erbB homology mapped to three exons that encode amino acid sequences having homologies of 64% and 67% to contig-uous regions within the tyrosine kinase domains of the EGFiR and erbB-2 proteins, respectively. A probe derived from the genomic DNA clone identified cDNA clones of the related mRNA which encode a predicted 148 kc~ transmembrane polypeptide with structural features identifying it as a member of the erbB family, prompting designation of the to new gene as erbB-3. This gene was mapped to human chromo-some 12q11-13 and was shown to be expressed as a 6.2 kb transcript in a variety of normal tissues of epithelial origin. Markedly elevated erbB-3 mRNA levels were demon-strated in certain human 'tumor cell lines.
Accordingly, in a principal embodiment, the present invention relates to a DNA segment having a nucleotide sequence that: encodes an erbB-3 gene or a unique portion thereof. This portion of an erbB-3 gene includes at least about 12 to 14 nucleotides which are sufficient to allow formation of a stable duplex with a DNA or RNA segment having sequences complementary to those in this portion of an erbB-3 gene. Further, this unique portion of an erbB-3 gene, of course, has a sequence not present in an erbB or an erbB-2 gene. In other words, the sequence of this portion of an erbB-3 gene differs in at least one nucleotide from the sequence of any other DNA
segment. In one embodiment, this DNA segment is exempli-fied by a human genomic DNA fragment that is produced by cleavage with the SacI restriction enzyme, has a size of about 90 kbp, and is detectable by nucleic acid hybridiza-tion with a probe derived from the v-erbB gene only under reduced stringency hybridization conditions, as described in Example 1. By application of the nucleic acid hybrid-ization and cloning methods described in the present disclosure, without undue: experimentation, one of ordinary skill in the art of recombinant DNA is enabled to identify and isolate DNA fragment:c related to the present human DNA
fragment comprising a nucleotide sequence that encodes at . 2t1 6990 0 .,~.WO 91/08214 PCT/US90/07025 least a portion of a mammalian erbB-3 gene other than the human erbB-3 gene. Application of the genomic DNA frag-ment of the erbB-3 gene as a probe in hybridization methods also enables one of ordinary skill in the art to obtain an entire erbB-3 gene, by sequential isolation of overlapping fragments adjoining the present fragment, i . a . , by an approach knowr,~ in the art as chromosome walking .
The present disclosure describes the partial nucleotide sequence of the human genomic 9 kbp SacI DNA
fragment, within the region of homology of the v-erbB
gene; however, the methods in the present disclosure further enable the iso7.ation and determination of the sequence of the entire !~ kbp human genomic DNA fragment according to the present invention. Accordingly, the Present invention further relates to a DNA segment having - the nucleotide sequence, or a unique portion thereof, of a human genomic DNA fragment that is produced by cleavage with the SacI restriction enzyme, has a size of about 9 kbp, and is detectable bay nucleic acid hybridization with a probe derived from the v-erbB gene only under reduced stringency hybridization conditions, as described in Example 1. By extension of the chromosome walking ap-proach noted above, the present invention further enables one of ordinary skill in the art to determination of the sequences of related DNA fragments comprising the complete human~erbB-3 gene as well, as erbB-3 genes of, for example, mammals other than human.
In the application of the present SacI DNA frag ment or any portion the=:eof as a probe for nucleic acid hybridization, the fragment is amplified, for example, by the in vitro polymerase chain reaction method (PCR; see U.S. Patent 4,683,202; U.S. Patent 4,683,195; and Saiki et al., 1985, Science 230:1:350-54) or by standard methods of molecular cloning. For example, a clone of the human erb8-3 gene DNA segment according to the present invention is exemplified by a recombinant clone of a normal human thymus DNA fragment, herein designated as the E3-1 genomic clone, having the partial restriction enzyme map defined .20 6 919 0 0 -~"~~O 91 /08214 ' a - PCT/US90107025 in Figure 2 and the partial DNA sequence defined in Figure 3 of the present application. Isolation and characteriza-v tion of genomic clone E3-1 is described in Example 2, below.
Analysis of the nucleotide sequences of the human geno~nic DNA segment according to the present invention reveals that the nucleotide sequence encodes three open reading frames bordered by splice junction consensus sequences which define the boundaries between non-translated intron sequences and the translated exons (Fig.
2). The predicted amino acid sequences of the three exons are highly similar to three regions which are contiguous in the tyrosine kinase domains of V-erbB, as well as human ~ EGF-R and erbB-2 proteins. Moreover, the predicted amino acid sequences of this human genomic clone are included in a larger open reading frame in complementary DNA (cDNA) clones of an mRNA species that is detected by hybridiza tion of a probe derived from the human genomic DNA clone.
Accordingly, the present invention also relates to a DNA segment having a nucleotide sequence of an erbB-3 gene in which that nucleotide sequence encodes the amino acid sequence of an erbH~-3 gene or a unique portion thereof. In other words, t:he sequence of this portion of an erbB-3 amino acid sequence differs in at least one wino acid residue from the amino acid sequence encoded by any other DNA segment. This portion of an erbB-3 amino acid sequence includes at least about 4 to 6 amino acids which are sufficient to provide a binding site for an antibody specific for this portion of the erbB-3 polypep-tide. Further, this unique portion of an erbB-3 amino acid sequence, of course, includes sequences not present in an erbB or an erbB-2 gene. In particular, the present invention relates to such a DNA segment for which this amino acid sequence or unique portion thereof is that of the polypeptide product of the human erbB-3 gene. This DNA segment is exemplified by the human genomic DNA clone E3-1, above, as well as by human cDNA clones designated E3-6, E3-8, E3-9, E3-11 and E3-16, which are described in C

' 9 ' PCT/US90/07025 vV0 9l /08214 Example 3 below. A preferred embodiment of this DNA
segment that encodes the aunino acid sequence of the entire polypeptide product of the: human erbB-3 gene is human cDNA
clone E3-16 having the nucleotide sequence defined in Figure 4 and having the predicted amino acid sequence defined in Figure 4.
The DNA segments according to this invention are useful for detection of expression of erbB-3 genes in normal and tumor tissues, as described in Example 5 below.
Therefore, in yet another aspect, the present invention relates to a bioassay f'or detecting erbB-3 mRNA in a biological sample comprising the steps of: i) contacting that biological sample with a DNA segment of this inven-tion under conditions such that a DNA: RNA hybrid molecular containing this DNA segmesnt and complementary RNA can be formed; and ii) determining the amount of that DNA segment present in the resulting hybrid molecule. Findings described in Example 5, below, indicate that increased erbB-3 expression, as detected by this method of this 2o invention, plays a role i.n some human malignancies, as is the case for the EGF-R (erbB) and erbB-2 genes.
Of course, it will be understood by one skilled in the art of genetic engineering that in relation to produc-tion of erbB-3 polypeptid~e products, the present invention 25 also includes DNA segments having DNA sequences other than those in the present examples that also encode the amino acid sequence of the po:lypeptide product of an erb8-3 gene. For example, it is known that by reference to the universal genetic code, standard genetic engineering 30 methods can be used to produce synthetic DNA segments having various sequences that encode any given amino acid sequence. Such synthetic DNA segments encoding at least a portion of the amino acid sequence of the polypeptide product of the human erbB-3 gene also fall within the 35 scope of the present invention. Further, it is known that different individuals may have slightly different DNA
sequences for any given human gene and, in some cases, such mutant or variant genes encode polypeptide products __ T _ 2o s9so 0 ",CVO 9l /08214 - 10 - PCT/US90/07025 having amino acid sequences which differ among individuals without affecting the essential function of the polypep-tide product. Still further, it is also known that many amino acid substitutions can be made in a polypeptide product by genetic engineering methods without affecting the essential function of: that polypeptide. Accordingly, the present invention further relates to a DNA segment having a nucleotide sequence that encodes an amino acid sequence differing in at: least one amino acid from the l0 the present invention further relates to a DNA segment having a nucleotide sequence that encodes an amino acid sequence differing in at least one amino acid from the amino acid sequence of h~unan erbH-3, or a unique portion thereof, and having greater overall similarity to the amino acid sequence of human erbH-3 than to that of any other polypeptide. The amino acid sequence of this DNA
segment includes at least: about 4 to 6 amino acids which are sufficient to provides a binding site for an antibody specific for the portion of a polypeptide containing this sequence. In a preferred embodiment, this DNA segment encodes an amino acid sequence having substantially the function of the human erbB-3 polypeptide. As noted above, the predicted erbH-3 polypeptide is a 148 Kd transmembrane polypeptide with structural features identifying it as a member of the erbH receptor family.
The similarity off: the amino acid sequence of the present invention with that of an erbB-3 amino acid sequence is determined by the method of analysis defined by the sequence alignment and comparison algorithms 3o described by Pearson and Lipman (Pearson, W.R. & Lipman, D. J., 1988, Proc. Nat. Acad. Sci. U.S.A. 85:2444-48).
This comparison contemplates not only precise homology of amino acid sequences, but also substitutions of one residue for another which. are known to occur frequently in f~ilies of evolutionarily related proteins sharing a conserved function.
The present invention further relates to a recom-binant DNA molecule comprising DNA segment of this inven-,,-..W0 91!08214 _ 11 _ PCT/US90/07025 tion and a vector. In yet another aspect, the present invention relates to culture of cells transformed with a DNA segment according to this invention. These host cells transformed with DNAs of the invention include both higher eukaryotes, including animal, plant and insect cells, and lower eukaryotes, such as. yeast cells, as well as prokary otic hosts including bacterial cells such as those of E.
c~oli and Bacillus subtilis. These aspects of the inven tion are exemplified :by recombinant DNAs and cells described in Examples 2 <~nd 3 below.
One particular embodiment of this aspect of this invention comprises a ce:Ll, preferably a mammalian cell, transformed with a DNA. of the invention, wherein the transforming DNA is capable of being expressed to produce the functional polypepti.de of an erbB-3 gene. Far exam-ple, mammalian cells (COS-1) transformed with the pSV2 gpt vector carrying the E3-1.6 cDNA, are prepared according to well-known methods, such as those described in Pierce, J. H. et al., 1988, Science 239:628-631; and Matsu:i, T., Heidaran, M., Miki, T., Popescu, N., La Rochel:Le, W., Kraus, M., Pierce, J. &
Aaronson, S., 1989, Science 243:800-804). Brief !y, cDNA
expression plasmids are: constructed by introducing the erbB-3-related cDNA encompassing all the nucleotides in the open reading frame into the pSV2 gpt vector into which the simian sarcoma virus long-terminal-repeat (LTR) had been engineered as the promoter, as previously described in detail. Transient expression an erbB-3 gene in such recombinant vectors is achieved by transection into COS-1 cells.
Stable expression of an erb8-3 gene can also be obtained with mammalian expression vectors such as the pZIPNEOSVX vector (Cepko, C. L., Roberts, B.E. and Mulli-gan, R. C., 1984, Ce~!3 3?:1053-62). For example, a eukaryotic expression vector was engineered by cloning the full-length erbB-3 coding sequence derived from cDNA clone E3-16 into the BamHI site of the pZIPNEOSVX vector. DNA

- lla -adapting the DNA fragments with synthetic oligonu-cleotides. NIH3T3 cells were transfected with 1 ~g of recombinant expression vector DNA (LTRerbB-3) and selected with the resistance marker antibiotic 6418. To detect expression of erbB-3, a polyclonal rabbit antiserum was ""'191/08214 _ 12 _ PCT/US90/07025 raised against a synthetic peptide (amino acid positions 1191-1205) within the predicted carboxyl terminus of the erbB-3 coding sequence. As shown in Figure 8, immuno-blotting analysis led to detection of the erbB-3 protein (Fig. 8A). The specificity of erbB-3 protein detection was demonstrated by preincubating the antiserum with the homologous peptide (Fig. 8B). Moreover, the normal 180 kD
erbB-3 protein was specifically detected with the poly-clonal antiserum only in cells transfected with the recom-binant erbH-3 expression vector, while control NIH3T3 cells that were not transfected with the vector were negative. The stably transfected NIH3T3 cells are useful as erbB-3_receptor protein sources for testing potential candidates for an erbB-3--specific ligand, analysis of the biological activity, as well as generation of monoclonal antibodies raised againsvt the native erbB-3 protein. An erbB-3-specific ligand is identified by detection of autophosphorylation of the erbH-3 receptor protein, stimulation of DNA synthesis or induction of the trans-formed phenotype of th.e LTRerbB-3 transfected NIH3T3 cells.
Alternatively, other transformed cell systems are available f or functional. expression of receptors of the erbB receptor family, fox example, a system based on the 32D cell line, a mouse hematopoietic cell line normally dependent on interleukin--3 (I1-3) for survival and prolif-eration. Recent studie:c have established that introduc-tion of an expression vector for the EGF-R in these cells leads to effective coupling with EGF mitogenic signal transduction pathways, i~hereby allowing a ligand of the EGF-R to replace I1-3 in supporting survival and growth of the 32D .cells. By employing the known methods described for the EGF-R, for example (Pierce, J. H. et al., 1988, supra),, the E3-16 cDNA of the present invention is ex-Pressed to produce functional receptr-s in 32D cells which are then useful for examining the biologl..cal function of these erbB-3 receptors, for instance, the specificity of .-.. ' 20 X6990 0 their ligand binding capacity and coupling capacities to secondary messenger systems. Thus, by so using gene expression methods described herein with the DNAs of the present invention, especially the preferred E3-16 cDNA
clone, one of ordinary skill in the art, without undue experimentation, can construct cell systems which fall within the scope of this invention, for determining the mechani$ms of erbB-3 regulatory processes. Accordingly, the present invention also relates to a bioassay for testing potential analogs of ligands of erbB-3 receptors for the ability to affects an activity mediated by erbB-3 receptors, comprising t:,he steps of: i) contacting a molecule suspected of being a ligand with erb8-3 receptors produced by a cell producing functional erbB-3 receptors;

and ii) determining the amount of a biological activity mediated by those erbB-3 receptors.

Various standard recombinant systems, such as those cited above as well as others known in the art, are suitable as well for production of large amounts of the novel erbB-3 receptor protein using methods of isolation for receptor proteins that are well known in the art.

Therefore, the present invention also encompasses an isolated polypeptide having at least a portion of the amino acid sequence defimed in Figure 4.

This invention further comprises an antibody specific for a unique portion of the human erbB-3 polypep-tide having the amino acid sequence defined in Figure 4, or a unique portion thereof. In this embodiment of the invention, the antibodies are monoclonal or polyclonah-in origin, and are generated using erbB-3 receptor-related polypeptides or peptides from natural, recombinant or synthetic chemistry sources. These antibodies specifical-ly bind to an erbB-3 protein which includes the sequences of such polypeptide. In other words, these antibodies bind only to erbB-3 receptor proteins and not to erbB

(EGF-R) or erbB-2 proteins. Also, preferred antibodies of this invention bind to an erbB-3 protein when that protein is in its native (biologically active) conformation.

h ; 91 /08214 - .14 -Fragments of antibodies of this invention, such as Fab or F(ab)' fragments, which retain antigen binding activity and can be prepared by methods well known in the art, also fall within the scope of the present invention.

Further, this invention comprises a pharmaceutical compo-sition of the antibodies of this invention, or an active fragment thereof, which can be prepared using materials and methods for preparing pharmaceutical compositions for administration of polypeptides that are well known in the art and can be adapted ~:eadily for administration of the present antibodies without undue experimentation.

These antibodies and active fragments thereof, can be used, for example, for specific detection or purifica-tion of the novel erbB-3 receptor. Such antibodies could also be used in various methods known in the art for targeting drugs to tis.;ues with high levels of erbB-3 receptors, for example, in the treatment of appropriate tumors with conjugates o:E such antibodies and cell killing agents. Accordingly, the present invention further relates to a method for targeting a therapeutic drug to cells having high levels of erb8-3 receptors, comprising the steps of i ) conjugating an antibody specific for an erbB-3 receptor, or an active fragment of that antibody, to the therapeutic drug; and ii) administering the result-ing conjugate to an individual with cells having high levels of erbB-3 receptors in an effective amount and by an effective route such that the antibody is able to bind to the erbB-3 receptors on those cells.

The antibody of this invention is exemplified by 3o rabbit antisera containing antibodies which specifically bind to erbB-3 protein. Such receptor specific antisera are raised to synthetic peptides representing a unique portion of the erbB-3 amino acid sequence, having six or more amino acids in sESquences which are sufficient to provide a binding site for an antibody specific for this portion of the erbB-3 polypeptide. Further, this unique portion of an erbB-3 amino acid sequence, of course, includes sequences not present in an erbB or an erbH-2 .20 fi990 0 aV0 91 /08214 ~- 15 - PCT/US90/07025 amino acid sequence, as predicted by the respective cDNA
- I sequences. The erbB-3 specific anti-peptide antibody of th~ present invention is exemplified by an anti-peptide antibody in polyclonal rabbit antiserum raised against the synthetic peptide having the sequence (in single letter amino acid code) EDEDEE'YEYM,NRRRR representing amino acid positions 1191-1205 in tale predicted sequence of the erbB-3 polypeptide. The specific detection of erbB-3 polypep-tide with this antiserum; is illustrated in mammalian cells transformed with an expression vector carrying a human erbB-3 cDNA (see Figure; 8A and 8B).
Antibodies to ps:ptides are prepared by chemically synthesizing the peptid~as, conjugating them to a carrier protein, and injecting the conjugated peptides into rabbits with complete Freund's adjuvant, according to standard methods of peptide immunization. For example, the peptide is synthesized by standard methods (Merrifield, R. B., 19!i3, J. Amer. Soc., 85:2149) on a solid phase synthesizer. The crude peptide is purified by 2o HPLC and conjugated to the carrier, keyhole limpet hemocy-anin or bovine thyroglobulin, for example, by coupling the amino terminal cysteine to the carrier through a maleimido linkage according to well known methods (e.g., Lerner, R.
A. et al . , 1981, Proc. ,Nat. Acad. Sci. USA, 78:3403 ) . In one standard method of peptide immunology, rabbits are immunized with 100 ~g of the erbB-3 peptide-carrier conjugate (1 mg/ml) i.n an equal volume of complete Freund's adjuvant and then boosted at 10-14 day intervals with 100 ~g of con jugat:ed peptide in incomplete Freund' s adjuvant. Additional boosts with similar doses at 10-14 day intervals are continued until anti-peptide antibody titer, as determined, for example, by routine ELISA
assays, reaches a plateau.
Thus, by following the teachings of the present disclosure, including application of generally known immunological methods cited herein, one of ordinary skill in the art is able to obtain erbB-3-specific antibodies and use them in a var:lety of imanunological assays, for 20 fi990 0 ~ 91 /08214 ' _ 16 _ PCT/US90/07025 example, for diagnostic detection of unusually high or low expression in normal or tumor tissues. Thus, the present invention also relates to a bioassay for detecting an erbB-3 antigen _. a biological sample comprising the steps of: i) contacting that sample with an antibody of the present invention specific for an erbB-3 polypeptide, under conditions such that a specific complex of that antibody and that antigen c:an be formed; and ii) determin-ing the amount of that antibody present in the form of those complexes.
The present invention may be understood mare readily by reference to the: following detailed description of specific embodiments .and the Examples and Figures included therein.
DESCRIPTION OF SPECIFIC EMBODIMENTS
The identification of a third member of the erbBEGF receptor family of membrane spanning tyrosine kinases and the cloning of its full length coding sequence 2o is described in the Examples herein. The presence of apparent structural domains resembling those of the EGF
receptor suggests the existence of an extracellular binding site for a ligand. The structural relatedness of the extracellular domain of the erbB-3 receptor with that of the EGF receptor indi<:ates that one or more of an increasing number of EGF-like ligands (Shoyab, M., Plow man, G. D., McDonald, V. L., Bradley, J. G. & Todaro, G.
J., 1989, Science 243:1074-1076) interacts with the erbB-3 product. Accordingly, the erbB-3 gene is expected to play important roles in both normal and neoplastic processes, , as is known for the EGF-R a.nd erb8-2 genes.
Despite extensive collinear homology with the EGF
receptor and erbB-2, distinct regions within the predicted erbB-2, coding sequence revealed relatively higher degrees of divergence. For examplE~, its carboxyl terminal domain failed to exhibit significant collinear identity scores with either erbB-2 or EGF-R. The divergence at the .,.CVO 91/08214 PC'T/US90/07025 carboxyl terminus also accounts for minor size differences among the three polypeptid~ss of erbB-3, erbB-2, and EGF-R, which possess estimated molecular weights of 148 kilo-daltons (kd), 138 kd, and 131 kd, respectively. Within the tyrosine kinase domain, which represents the most conserved region of the predicted erbB-3 protein, a short stretch of 29 amino acids closer to the carboxyl terminus than the ATP binding sited differed from regions of the predicted erbB-2 and EGF-R coding sequence in 28 and 25 positions, respectively. Such regions of higher diver-gence in their cytoplasmic domains are likely to confer different functional specificity to these closely related receptor-like molecule, Thus, mutations or other alter-ations in expression of the erbB-3 gene are likely to cause cancers or genetic disorders different from those associated with such defects in the erbB and erbB-2 genes.
Chromosomal mapping localized erbB-3 to human chromosome 12 at the q11-13 locus, whereas the related EGF-R and erbB-2 genes are located at chromosomal sites 7p12-13 and 17p12-21.3, respectively. Thus, each gene appears to be localized to a region containing a different homeobox and a different collagen chain gene locus.
Keratin type I and type II genes also map to regions of 12 and 17, respectively, consistent with the different localizations of erbB-3 and erbB-2, respectively. Thus, the DNA segments of the present invention represent novel probes to aid in genetic mapping of any heritable diseases which are associated with. chromosomal aberrations in the vicinity of the 12q11-13 7_ocus.
There is evidence for autocrine as well as para-crine effectors of normal cell proliferation. The former are factors that are produced by the same cells upon which they stimulate cell proliferation, whereas the latter factors are secreted by dwells other than those that are affected by those factors. However, the inherent trans-forming potential of aut:ocrine growth factors suggests that growth factors most commonly act on their target cell populations by a paracrir~e route . The present survey of PCT/US90/07~-..,"S
WO 9l /08214 IerbB-3 gene expression indicates its normal expression in cells of epithelial and neuroect~ermal derivation. ' Comparative analysis of ths: three erbBt receptor-like genes in different cell types of epidermal tissue revealed that keratinocytes expressed all three genes. In contrast, melanocytes and stromal f:ibroblasts specifically lacked EGF-R and erbB-3 transcripts, respectively. Thus, melano-cytes and stromal fibrobla.sts may be sources of paracrine growth factors for EGF-R and erbB-3 products, respective-1y, that are expressed by the other cell types residing in close proximity in epidermal tissues.
Given that both ezbB and erbB-2 have been casually implicated in human malignancy, the present findings (Example 5) that the erbB-3 transcript is overexpressed in a significant fraction of human mammary tumor cell lines indicates that this new member of the EGF-R receptor family also plays an important role in some human malig-nancies.
_Example 1. Ident:Lf ication of a human DNA f racxment related to the erbB uroto-oncoctene family. In an effort to detect novel erbB-related genes, human genomic DNA was cleaved with a variety of restriction endonucleases and subjected to Southern blot analysis with v-erbB as a probe. Normal mammary epithelial cells AB589 (Walen, K.
H. & Stampfer, M. R., 1989, Cancer. Genet. Cytogenet.
37:249-261) and immortalized keratinocytes RHEK have been described previously (Rhim, J. S., Jay, G., Arnstein, P., Price, F. M., Sanford, K. K. & Aaronson, S. A., 1985, Science 227:1250-52). Normal human epidermal melanocytes (NHEM) and keratinocytes (NHEK) were obtained from Clonetics. Sources for human embryo fibroblasts (Rubin, J . S . , Osada, H . , Finch, P . W . , Taylor, W . G . , Rudikof f , S., & Aaronson, S. A., 1989, Proc. Nat. Acad. Sci. USA
86:802-806) or mammary tumor cell lines SK-BR-3, MDA-MB468, MDA-MB453, and MD.A-MB415 (Kraus, M. H., Popescu, N.
C., Amsbaugh, S. C. & R:ing, C. R., 1987 EM90. J. 6:605-610) have been described. For nucleic acid RNA hybridiza-tion, DNA and RNA wex:e transferred to nitrocellulose ,~-. WO 91 /08214 PCT/US90/07025 19 - ~~~99~0 membranes as previously described (Kraus, K. H., et al., 1987, supra). High stringency hybridization was conducted in 50~ formamide and 5xSSC at 42°C. Filters were washed at 50°C in 0.lxSSC. Reduced stringency hybridization of DNA was carried out in 30~ formamide followed by washes in 0.6xSSC, while intermediate stringency was achieved by hybridization in 40~ formamide and washing in 0.25xSSC.
For the specific results depicted in Fig. 1, DNAs were restricted with SacI and hybridized with probe specific for an oncogenic viral form of the erbB gene, v-erbB, spanning from the upstream BamHI site to the EcoRI site in the avian erythroblastosis proviral DNA (Vennstrom, B., Franshier, L., Moscovici, C. & Bishop,~~:J. M., 1980, J.
Virol. 36:575-585).
Under reduced stringency hybridization, four SacI
restriction fragments were detected. Two were identified as EGF-R gene fragments by their amplifications in the mammary tumor cell line MDA-MB468 (Fig. 1A, lane 1,2) known to contain EGF-R gene amplification and one as an erbB-2 specific gene fragment due to its increased signal intensity in another mammary tumor cell line, SK-BR-3, known to have erbB-2 amplified (Fig. 1A, lane 1,3).
However, a single 9 kbp SacI fragment exhibited equal signal intensities in DNAs from normal human thymus, SK-~BR-3 and a line with high. levels of EGF-R, A431 (Fig. 1A).
When the hybridization stringency was raised by 7°C, this fragment did not hybridize, whereas EGF-R and erbB-2 specific restriction fragments were still detected with v-erbB as a probe (Fig. 1B). Taken together, these findings suggested the specific detection of a novel v-erbB-related DNA sequence within the 9 kbp SacI fragment.
Example 2.' Claninc~ of the human DNA fragment related to erbB. For further characterization a normal human genomic library was prepared from SacI cleaved thymus DNA enriched for 8 to 12 kbp fragments. For convenience, bacteriophage ~,sep6-lacy was obtained from L.
Prestidge and D. Hogness (Stanford University); many other cloning vectors derived from phage ~, or other genomes can O 91 /08214 ~ ~ 6 9 9 be used for cloning this :DNA fragment according to stan-dard recombinant DNA methods that are well known in the art. Purified phage DNA was subjected to cos-end liga-tion, restriction with SacI, and fractionation in a continuous 10-40~ sucrose gradient. A genomic library was prepared by ligating Sacl restriction fragments of normal human thymus DNA in the molecular weight range of 8 kbp to 12 kbp (isolated by sucrose gradient sedimentation) with the purified phage arms. Ten recombinant clones detected by v-erbB under reduced stringency conditions did not hybridize with human EGF-R or erbB-2 cDNA probes at high stringency. As shown in the restriction map of a repre-sentative clone with a 9 l~:bp insert, the region of v-erbB
homology was localized by hybridization analysis to a 1.5 kbp segment spanning from the EcoRI to the downstream PstI
site.
The nucleotide sequence of a portion of a clone of the novel human genomic DNA fragment related to erb8 was determined for both DNA strands by the dideoxy chain termination method (Sanger, F., Nicklen, S. & Coulson, A.
R., 1977, Proc. Nat. Acad. Sci. USA. 74:5463-67) using supercoiled plasmid DNA as template. The nucleotide ~quence was assembled and translated using IntelliGenetics software. Amino acid sequence comparison was performed with the alignment program by Pearson and Lipman (Pearson, W. R. & Lipman, D. J., 1988, supra) as implemented on the computers of the NCI Advanced Scientif-ic Computing Laboratory. Hydrophobic and hydrophilic regions in the predicted protein were identified according to Ryte and Doolittle (Ryte, J. & Doolittle, R. F., 1982, J. Mol. Biol. 157:105-132). Nucleotide sequence analysis revealed that the region of v-erbB homology in the 1.5 kbp segment from the EcoRI to the PstI contained three open reading frames bordered by splice junction consensus sequences (Fig. 3). Computerized comparisons of the predicted amino acid sequence of these three open reading frames with other known proteins revealed the highest identity scores of 64$ to~ 67$ to three regions which are v, ~mosZ~a 2 0 0 9 9 0 0 r. P~'~US90/07025 contiguous in the tyrosine kinase domains of v-erbB, as well as human EGF-R and erbB-2 proteins. Furthermore, all splice junctions of the three characterized exons in the new gene were conserved with erbB-2. Amino acid sequence homology to other known tyrasine kinases was significantly lower, ranging from 39% to ~6%.
A single 6.2 kb specific mRNA was identified by Northern blot analysis of human epithelial cells using the 150 by SpeI-AccI exon-containing fragment as probe (Fig.
2). Under the stringent hybridization conditions em ployed, this probe detected neither the 5 kb erbB-2 mRNA
nor the 6 kb and 10 kb EGF-R mRNAs. All of these findings suggested that the present work has identified a new functional member of the erbB proto-oncogene family, which tentatively has been designated as erbB-3.
Example 3. Clonina~ and characterization of cDNAs f or the mRNA of the human erbB-3 Qene . In an ef fort to characterize the entire erbB-3 coding sequence, overlap-ping cDNA clones were isolated from oligo dT-primed cDNA
libraries from sources with known erbB-3 expression, utilizing gene-specific genomic exons or cDNA fragments as probes. In brief, an oliga dT-primed human placenta cDNA
library in ~gtll was obtained from Clontech. MCF-7 cDNA
was prepared by first strand synthesis from 5 ~g poly A' RNA using an oligo dT conta.ining linker-primer and Mo-MuI~V
reverse transcriptase, followed by second strand synthesis with DNA polymerase I, RNaseH, and subsequent T4 DNA
polymerase treatment. Double-stranded cDNA was direction-ally cloned into the SfiI site of ~,pCEV9 using specific linker adapter oligonucleotides (Miki, T., Matsui, T., Heidaran, M. A. & Aaronson,, S. A., 1989, Gene 83:137-146 Following plaque purification, phage DNA inserts were subcloned into pUC-based plasmid vectors for further characterization. The clones were initially characterized :by restriction analysis and hybridization to the mRNA, and were subsequently subjected to nucleotide sequence analysis. Clones designated pE3-6, ..-a.~0 91/0$214 PCT/US90/07Q=
~- # , : - 22 -pE3-8, pE3-9, and pE3-11 carrying inserts with molecular weights ranging from 1.3 kpb to 4.3 kbp were isolated from a human placenta library, whereas the pE3-16 clone con-taining a 5 kbp insert was obtained by screening the MCF-7 cDNA library with the upstream most coding sequence of pE3-11 as a probe. The clones pE3-8, pE3-9, pE3-11, and pE3-16 contained identical 3' ends terminating in a poly A
stretch (Fig. 2).
The complete coding sequence of erbB-3 was con tained within a single long open reading frame of 4080 nucleotides extending from position 46 to an in-frame termination codon at position 4126. The most upstream ATG
codon at position 100 was the likely initiation codon, as it was preceded by an in-frame stop codon at nucleotide position 43 and fulfilled the criteria of Kozak for an authentic initiation codon. The open reading frame comprised 1342 codons predicting a 148 kd polypeptide.
Downstream from the termination codon, multiple stop codons were present in a7L1 frames. As shown in Fig. 5, the deduced amino acid sequence of the erbB-3 polypeptide predicted a transmembrane receptor tyrosine kinase most closely related to EGF-R and erbB-2. A hydrophobic signal sequence of erbB-3 was predicted to comprise the 19 a.mino-terminal amino acid residues. Cleavage of this signal sequence between glycine at position 19 and serine at position 20 would generate a processed polypeptide of 1323 amino acids with an estimated molecular weight of 145 kd.
A single hydrophobic membrane spanning domain encompassing 21 amino acids was identified within the coding sequence separating an extracellula.r domain of 624 amino acids from a cytoplasmic domain comprising 678 amino acids (Fig. 5).
The putative erbB-3 ligand-binding domain was 43~
and 45$ identical in amino acid residues with the predict ed erbB-2 and EGF-R protein, respectively. Within the extracellular domain, all 50 cysteine residues of the processed erbB-3 polypepti.de were conserved and similarly spaced when compared to the EGF-R and erbB-2. Forty-seven cysteine residues were organized in two clusters contain-.-,WO 91/08214 PCT/US90/07025 ing 22 and 25 cysteines :respectively, a structural hall-mark of this tyrosine kinase receptor subfamily (see, for example, Yamamoto, T., Ik~awa, S., Akiyama, T., Semba, K., Nomura, N., Miyajima, N., Saito, T. & Toyoshima, K., 1986, Nature 319:230-234). Ten potential N-linked glycosylation sites were localized within the erbB-3 extracellular domain. In comparison with the EGF-R and erbB-2 proteins, five and two of these glycosylation sites were conserved, respectively. Among these, the site proximal to the transmembrane domain was conserved among all three pro-teins (Fig. 5).
Within the cytoplasmic domain, a core of 277 amino acids from position 702 through 978 revealed the most extensive homology with the tyrosine 'kinase domains of EGF-R and erbB-2. In this. region 60~ or 62~ of amino acid residues were identical .and 90~ or 89$ were conserved, respectively. This stretch of amino acid homology coin-cides with the minimar catalytic domain of tyrosine kinases (Hanks, S. K., Quinn, A. M. & Hunter, T., 1988, Science 241:42-52). Theres was significantly lower homolo-gy with other tyrosine kinases (Fig. 5). The consensus sequence for an ATP-binding site GxGxxG (Hanks, S. K. et a~. , 1988, supra) was identified at amino acid positions 716 through 721. This sequence as well as a lysine residue located 21 amino acid residues further toward the carboxyl terminus were conserved between the three erbB-related receptors. Taken together these findings defined the region between amino acid position 702 and 978 as the putative catalytic domain of the erbB-3 protein (Fig. 5).
The most divergent region of erbB-3 compared to either EGF-R or erbB-2 wags its carboxyl terminus compris-ing 364 amino acids. This region showed a high degree of hydrophilicity and the frEaquent occurrence of proline and tyrosine residues. Among these tyrosine residues, those at positions 1197, 1199, and 1262 matched closest with the consensus sequence for putative phosphorylation sites.
The peptide sequence YEYMN, encompassing tyrosine 1197 and 1199, was repeated at posLtions 1260-1264 and was at both 4 PCT/US90/07~°-.

locations surrounded by charged residues, providing an environment of high local hydrophilicity. These observa tions render tyrosines 1197, 1199 and 1262 likely candidates for autophosphorylation sites of the erbB3 protein.
Example 4. Chromosomal mapping of the human erbB-3 gene. The chromosomal location of the erbB-3 gene was determined by in situ hybridization (Popescu, N. C., King, C. R. & Kraus, M. H., 1989, Genomics 4:362-366) with a 3H-labeled plasmid containing the amino-terminal erbB-3 coding sequence. A total of 110 human chromosome spreads were examined prior and subsequent to G banding for identification of individual chromosomes. A total of 142 grains were localized on a 400-band ideogram. Specific labeling of chromosome 12 was observed, where 38 out of 51 grains were localized to band q13 (Fig. 6). Thus, the genomic locus of erbB-3 was assigned to 12q13. In this region of chromosome 12, several genes have previously been mapped including the melanoma-associated antigen ME491, histone genes and the gene for lactalbumin. In addition, two proto-oncogenes, int-1 and g1i are located in close proximity to erbB-3.
Example 5. ErbB-3 exyression in normal and malicrnant human cells. To investigate its pattern of expression, a number of human tissues were surveyed for the erbB-3 transcript. The 6.2 kb erbB-3 specific mRNA
was observed in term placenta, postnatal skin, stomach, lung, kidney, and brain, while it was not detectable in skin fibroblasts, skeletal muscle or lymphoid cells.
Among the fetal tissues analyzed, the erbB-3 transcript was expressed in liver, kidney, and brain, but not in fetal heart or embryonic lung fibroblasts. These observa-tions indicate the preferential expression of erbB-3 in epithelial tissues and brain.
ErbB-3 expression was also investigated in indi-vidual cell populations derived from normal human epithe-lial tissues including keratinocytes, glandular epithelial cells, melanocytes, and fibroblasts. For comparison ..-..WO 91/08214 2 p 6 9 g 0 p PCT/US9a/07025 levels of EGF-R and erbB-2 transcripts were analyzed. As shown in Table 1, erbB-3 :mRNA levels were relatively high in keratinocytes, comparable with those of erbB-2 and EGF-R in these cells. Lower, but similar expression levels of each transcript were detected in cells derived from glandular epithelium. These findings are consistent with growth regulatory roles of all three receptor-like mole-cules in squamous and glandular epithelium. Whereas erbB-2 and EGF-R transcripts were also readily observed in normal fibroblasts, the same cells lacked detectable erbB-3 mRNA. In contrast, normal human melanocytes, which expressed both erbB-3 and erbB-2 at levels comparable with human keratinocytes, lacked: detectable EGF-R transcripts.
Thus, the expression p~~tterns of these receptor-like molecules were different in specialized cell populations derived from epidermal tissues.
Table 1: Normal expression pattern of human erbB gene family members.
Relative Cell Source of Transcript:; Gene RNA levels Embryonic fibroblast (M42fi) erbB-3 -erbB-2 +
EGF-R +
Skin fibroblast (501T) erbB-3 -erbB-2 +
EGF-R +
Immortal keratinocyte (RHEK) erbB-3 ++
erbB-2 ++
EGF-R ++
Primary keratinocyte (NHE1C) erbB-3 +
erbB-2 +
EGF-R ++
Glandular epithelium (AB5E39) erbB-3 (+) erbB-2 (+) EGF-R (+) Melanocyte (NHEM) erbB-3 ++
erbB-2 ++
EGF-R -~. O 91 /08214 , 2 0 6 9 9 0 0 PCT/US90/07025 Replicate Northern blot., were hybridized with equal amounts (in cpm) of probe;; of similar specific activities for erbB-3, erbB-2, and EGF-R, respectively. Relative signal intensities were estimated: - not detectable, (+) weakly positive, + positive, ++ strongly positive.
To search for ev:Ldence of erbB-3 involvement in the neoplastic process, erbB-3 mRNA levels in a series of human tumor cell lines ware surveyed. The erbB-3 tran-script was detected in 36 of 38 carcinomas and 2 of 12 sarcomas while 7 tumor cell lines of hematopoietic origin lacked measurable erbB-3 mRNA. Markedly elevated levels of a normal-sized transcript were observed in 6 out of 17 tumor cell lines derived from human mammary carcinomas.
By Southern blot analysis, neither gross gene rearrange-ment nor amplification was detected in the cell lines.
Figure 7A shows the results of Northern blot analysis with control AB589 nonmalignant human mammary epithelial cells (lane 1) and two representative human mammary tumor lines, MDA-MB415 (lane 2) and MI)A-MB453 (lane 3). Hybridization of the same filter with a human ~-actin probe (Fig. 7B) verified actual levels of mRNA in each lane. Densito-metric scanning indicated that the erbB-3 transcript in each tumor cell line was elevated more than 100 fold above that of the control cel7L line. Thus, overexpression of this new member of the e:rbB family, as in the case of the EGF-R and erbB-2 genes, is likely to play an important role in some human malignancies.
The foregoing invention has been described in some detail for purposes of c7.arity and understanding. It will ', a,' -.WO 91/08214 PCT/US90/07025 _ 2 7 _ '~-~~ y' ~ g-.0 ~
also be obvious that various changes and combinations in form and detail can be :made without departing from the scope of the invention.

Claims (27)

1. A DNA segment having a nucleotide sequence that encodes an erbB-3 polypeptide or a portion thereof sufficient to provide an erbB-3 receptor protein binding site for an antibody thereto, said nucleotide sequence consisting essentially of an at least 12 nucleotide portion of the sequence set for in Figure 4, or a sequence substantially identical thereto, wherein the nucleotide sequence portion is not present in an erbB gene or an erbB-2 gene.
2. The DNA segment according to claim 1, wherein said nucleotide sequence is a mammalian erbB-3 gene.
3. The DNA segment according to claim 2, wherein said mammalian gene is a human erbB-3 gene.
4. A DNA segment or a portion thereof for use as an erbB-3 specific probe, wherein said DNA segment has the nucleotide sequence of a genomic DNA fragment that is produced by cleavage with the Sac 1 restriction enzyme, has a size of about 9 kbp, and is detectable by nucleic acid hybridization with a probe derived from the v-erbB gene only under reduced stringency hybridization conditions, wherein said DNA
segment has the partial restriction enzyme map defined in Figure 2 and the partial DNA
sequence defined in Figure 3, and wherein said DNA segment or said portion thereof has a nucleotide sequence not present in an erbB gene or an erbB-2 gene.
5. The DNA segment according to claim 1, wherein said polypeptide sequence is that defined in Figure 4.
6. The DNA segment according to claim 5, comprising human cDNA clone E3-16 having the nucleotide sequence defined in Figure 4.
7. A DNA segment comprising a nucleotide sequence that encodes an amino acid sequence of Figure 4 or a portion thereof to provide an erbB-3 receptor protein binding site for an antibody thereto, which antibody does not bind an erbB-2 receptor protein or an erbB
receptor protein.
8. The DNA segment according to claim 7 that encodes an amino acid sequence having substantially the function of the human erbB-3 polypeptide.
9. An isolated polypeptide having an amino acid sequence encoded by the DNA
segment according to claim 7.
10. A recombinant DNA molecule comprising the DNA segment of claim 1 and a vector.
11. A culture of cells transformed with the DNA segment according to claim 1.
12. An isolated polypeptide having the amino acid sequence defined in Figure 4 or a portion thereof which is sufficient to provide a binding site for an antibody that specifically binds an erbB3 receptor protein, which antibody does not bind an erbB-2 receptor protein or an erbB receptor protein.
13. A bioassay for detecting erbB-3 nucleic acid in a biological sample comprising the steps of:
i) contacting said biological sample with a DNA segment according to claim 1 under conditions such that hybridization can occur; and ii) detecting the presence of hybridization, whereby the presence of hybridization detects erbB-3 nucleic acid in the biological sample.
14. A bioassay for identifying a ligand of an erbB-3 receptor, comprising the steps of:

i) contacting an erbB-3 receptor encoded by the DNA segment of claim 1 with a candidate ligand of an erbB-3 receptor; and ii) measuring an amount of biological activity mediated by the erbB-3 receptor contacted by the candidate ligand of an erbB-3 receptor, whereby a change in biological activity, relative to the amount of biological activity mediated by an erbB-3 receptor not contacted with the candidate ligand of an erbB-3 receptor, identifies a ligand of an erbB-3 receptor.
15. An antibody or an active fragment thereof that specifically binds an erbB-polypeptide having the amino acid sequence defined in Figure 4, or a sequence substantially identical thereto, wherein the antibody or the active fragment thereof does not specifically bind an erbB-2 polypeptide or an erbB polypeptide.
16. A bioassay for detecting an erbB-3 antigen in a biological sample comprising the steps of:
i) contacting said sample with an antibody according to claim 15, under conditions such that an antigen/antibody complex can be formed; and ii) detecting the presence of said complex, whereby the presence of said complex detects an erbB-3 antigen in said sample.
17. The use of an effective amount of a conjugate of an antibody according to claim 15, or an active fragment thereof, and a therapeutic drug for targeting cells having high levels of erbB-3 receptors by an effective route such that said antibody is able to bind to said erbB-3 receptors on said cells.
18. An isolated erbB-3-specific nucleic acid probe that hybridizes to at least part of a nucleic acid encoding an erbB-3 protein and does not hybridize to a nucleic acid encoding an erbB-2 protein or a nucleic acid encoding an erbB protein under high stringency hybridization conditions, wherein the erbB-3 protein has the amino acid sequence shown in Figure 4.
19. A recombinant DNA molecule comprising the nucleic acid of claim 18 and a vector.
20. A culture of cells transformed with the nucleic acid according to claim 18.
21. A bioassay for detecting erbB-3 nucleic acid in a biological sample comprising the steps of:
i) contacting said biological sample with a nucleic acid according to claim 18 under conditions such that hybridization can occur; and ii) detecting the presence of hybridization, whereby the presence of hybridization detects erbB-3 nucleic acid in the biological sample.
22. A culture of cells transformed with the recombinant DNA molecule of claim 10 or 19.
23. The antibody according to claim 15, wherein said antibody is a polyclonal antibody.
24. A composition comprising the antibody of claim 15, or an active fragment thereof, and a diluent, carrier or additive.
25. A composition comprising a conjugate of an antibody according to claim 15, or an active fragment thereof, and a cell killing agent, and a diluent, carrier or additive.
26. The use according to claim 17, wherein the conjugate kills the cells.
27. The antibody of claim 15, or an active fragment thereof, wherein the antibody or the active fragment thereof is coupled to a detectable label.
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