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

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 a 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.

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 spanning 
tyrosine 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 recognized 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 receptors in several of these 
families play critical roles in regulation of normal growth and 
development. Recent studies in DrosophiIa 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 being allelic 
to the zygotic embryonic lethal faint little ball exhibiting a complex 
phenotype with deterioration of multiple tissue components of ectodermal 
origin. Furthermore, 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 the 
DrosophiIa counterpart of both known mammalian erbB-related genes. 
Some of these receptor molecules have been implicated in the neoplastic 
process as well. In particular, both the erbB and erbB-2 genes have been 
shown to be activated as oncogenes by mechanisms involving overexpression 
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., Kraus, M. H., 
Segatto, O., King, 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 causally 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., 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. 
SUMMARY OF THE INVENTION 
It is an object of 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 whether 
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 epidermal growth factor 
receptor (EGF-R) (i.e., the erbB gene) and from the related erbB-2 gene. 
Characterization of this DNA fragment after partial 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 contiguous regions within the tyrosine kinase domains of the EGF-R 
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 kd transmembrane polypeptide with structural features identifying it 
as a member of the erbB family, prompting designation of the new gene as 
erbB-3. This gene was mapped to human chromosome 12qll-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 demonstrated 
in certain human mammary 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 exemplified by 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 gen only 
under reduced stringency hybridization conditions, as described in Example 
1. By application of the nucleic acid hybridization 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 fragments related to the present human DNA fragment comprising 
a nucleotide sequence that encodes at least a portion of a mammalian 
erbB-3 gene other than the human erbB-3 gene. Application of the genomic 
DNA fragment 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.e., by an approach known 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 to 
the v-erbB gene; however, the methods in the present disclosure further 
enable the isolation and determination of the sequence of the entire 9 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 by 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 approach 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 fragment or any portion thereof 
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. 
Pat. No. 4,683,202; U.S. Pat. No. 4,683,195; and Saiki et al., 1985, 
Science 230:1350-54) or by standard methods of molecular cloning. For 
example, a clone of the human erbB-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 in FIG. 2 and the partial DNA 
sequence defined in FIG. 3 of the present application. Isolation and 
characterization of genomic clone E3-1 is described in Example 2, below. 
Analysis of the nucleotide sequences of the human genomic 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 nontranslated 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 
hybridization 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 erbB-3 gene or a unique portion 
thereof. In other words, the sequence of this portion of an erbB-3 amino 
acid sequence differs in at least one amino 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 polypeptide. 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 Example 3 below. A preferred embodiment of this DNA 
segment that encodes the amino 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 FIG. 4 (A-L) and having the predicted amino 
acid sequence defined in FIG. 4 (A-L). 
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 for detecting erbB-3 mRNA in a biological sample 
comprising the steps of: i) contacting that biological sample with a DNA 
segment of this invention under conditions such that a DNA:RNA hybrid 
molecule containing this DNA segment 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 invention, plays a role in 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 production of erbB-3 polypeptide products, 
the present invention also includes DNA segments having DNA sequences 
other than those in the present examples that also encode the amino acid 
sequence of the polypeptide product of an erbB-3 gene. For example, it is 
known that by reference to the universal genetic code, standard genetic 
engineering 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 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 having amino acid sequences which differ among 
individuals without affecting the essential function of the polypeptide 
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 amino acid sequence of human erbB-3, or a 
unique portion thereof, and having greater overall similarity to the amino 
acid sequence of human erbB-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 provide 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 erbB-3 polypeptide is a 148 kd transmembrane 
polypeptide with structural features identifying it as a member of the 
erbB receptor family. 
The similarity of 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 
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 families of 
evolutionarily related proteins sharing a conserved function. 
The present invention further relates to a recombinant DNA molecule 
comprising a DNA segment of this invention 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 prokaryotic hosts including bacterial cells such as those of E. 
coli and EaciIIus subtilis. These aspects of the invention are exemplified 
by recombinant DNAs and cells described in Examples 2 and 3, below. 
One particular embodiment of this aspect of this invention comprises a 
cell, preferably a mammalian cell, transformed with a DNA of the 
invention, wherein the transforming DNA is capable of being expressed to 
produce the functional polypeptide of an erbB-3 gene. For example, 
mammalian cells (COS-1) transformed with the pSV2 gpt vector carrying the 
E3-16 cDNA are prepared according to well-known methods, such as those 
described in U.S. Pat. Application 07/308,302 of Matsui et al., filed Feb. 
9, 1989; see also Pierce, J. H. et al., 1988, Science 239:628-631; and 
Matsui, T., Heidaran, M., Miki, T., Popescu, N., La Rochelle, W., Kraus, 
M., Pierce, J. & Aaronson, S., 1989, Science 243:800-804). Briefly, 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 erbB-3 gene can also be obtained with mammalian 
expression vectors such as the pZIPNEOSVX vector (Cepko, C. L., Roberts, 
B. E. and Mulligan, R. C., 1984, Cell 37:1053-62). 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 adapting the DNA fragments with synthetic 
oligonucleotides. NIH3T3 cells were transfected with 1 .mu.g of 
recombinant expression vector DNA (LTRerbB-3 ) and selected with the 
resistance marker antibiotic G418. To detect expression of erbB-3, a 
polyclonal rabbit antiserum was raised against a synthetic peptide (amino 
acid positions 1191-1205) within the predicted carboxyl terminus of the 
erbB-3 coding sequence. As shown in FIG. 8, immunoblotting analysis led to 
detection of the erbB-3 protein (panel A). The specificity of erbB-3 
protein detection was demonstrated by preincubating the antiserum with the 
homologous peptide (panel B). Moreover, the normal 180 kD erbB-3 protein 
was specifically detected with the polyclonal antiserum only in cell 
transfected with the recombinant erbB-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 against the native erbB-3 protein. An erbB-3-specific 
ligand is identified by detection of autophosphorylation of the erbB-3 
receptor protein, stimulation of DNA synthesis or induction of the 
transformed phenotype of the LTRerbB-3 transfected NIH3T3 cells. 
Alternatively, other transformed cell systems are available for functional 
expression of receptors of the erbB receptor family, for example, a system 
based on the 32D cell line, a mouse hematopoietic cell line normally 
dependent on interleukin-3 (Il-3) for survival and proliferation. Recent 
studies have established that introduction of an expression vector for the 
EGF-R in these cells leads to effective coupling with EGF mitogenic signal 
transduction pathways, thereby allowing a ligand of the EGF-R to replace 
Il-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 expressed to 
produce functional receptors in 32D cells which are then useful for 
examining the biological function of these erbB-3 receptors, for instance, 
the specificity of 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 mechanisms 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 affect an activity mediated by erbB-3 receptors, comprising 
the steps of: i) contacting a molecule suspected of being a ligand with 
erbB-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 defined in FIG. 4. 
This invention further comprises an antibody specific for a unique portion 
of the human erbB-3 polypeptide having the amino acid sequence defined in 
FIG. 4 (A-L), or a unique portion thereof. In this embodiment of the 
invention, the antibodies are monoclonal or polyclonal in origin, and are 
generated using erbB-3 receptor-related polypeptides or peptides from 
natural, recombinant or synthetic chemistry sources. These antibodies 
specifically bind to an erbB-3 protein which includes the sequence 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. 
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 composition 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 readily for administration of the present 
antibodies without undue experimentation. 
These antibodies and active fragments thereof, can be used, for example, 
for specific detection or purification of the novel erbB-3 receptor. Such 
antibodies could also be used in various methods known in the art for 
targeting drugs to tissues with high levels of erbB-3 receptors, for 
example, in the treatment of appropriate tumors with conjugates of 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 erbB-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 resulting 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 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 sequences 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 erbB-2 amino acid sequence, as 
predicted by the respective cDNA sequences. The erbB-3 specific 
anti-peptide antibody of the 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) 
EDEDEEYEYMNRRRR representing amino acid positions 1191-1205 in the 
predicted sequence of the erbB-3 polypeptide. The specific detection of 
erbB-3 polypeptide with this antiserum is illustrated in mammalian cells 
transformed with an expression vector carrying a human erbB-3 cDNA (see 
FIG. 8). 
Antibodies to peptides are prepared by chemically synthesizing the 
peptides, 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., 1963, J. 
Amer. Soc., 85:2149) on a solid phase synthesizer. The crude peptide is 
purified by HPLC and conjugated to the carrier, keyhole limpet hemocyanin 
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. 
U.S.A., 78:3403). In one standard method of peptide immunology, rabbits 
are immunized with 100 .mu.g of the erbB-3 peptide-carrier conjugate (1 
mg/ml) in an equal volume of complete Freund's adjuvant and then boosted 
at 10-14 day intervals with 100 .mu.g of conjugated 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 variety of immunological assays, for 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 in 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 can be formed; and ii) determining the 
amount of that antibody present in the form of those complexes. 
The present invention may be understood more 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 erbB-EGF receptor family of 
membrane spanning tyrosine kinases and the cloning of its full length 
coding sequence 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 indicates that one or more of an increasing 
number of EGF-like ligands (Shoyab, M., Plowman, 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 and erbB-2 genes. 
Despite extensive collinear homology with the EGF receptor and erbB-2, 
distinct regions within the predicted erbB-3 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 carboxyl terminus also 
accounts for minor size differences among the three polypeptides of 
erbB-3, erbB-2, and EGF-R, which possess estimated molecular weights of 
148 kilodaltons (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 site differed from regions of the 
predicted erbB-2 and EGF-R coding sequence in 28 and 25 positions, 
respectively. Such regions of higher divergence in their cytoplasmic 
domains are likely to confer different functional specificity to these 
closely related receptor-like molecules. Thus, mutations or other 
alterations 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 qll-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 12qll-13 locus. 
There is evidence for autocrine as well as paracrine 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 cells other than those that are affected by those 
factors. However, the inherent transforming potential of autocrine growth 
factors suggests that growth factors most commonly act on their target 
cell populations by a paracrine route. The present survey of erbB-3 gene 
expression indicates its normal expression in cells of epithelial and 
neuroectodermal derivation. Comparative analysis of the three erbB 
receptor-like genes in different cell types of epidermal tissue revealed 
that keratinocytes expressed all three genes. In contrast, melanocytes and 
stromal fibroblasts specifically lacked EGF-R and erbB-3 transcripts, 
respectively. Thus, melanocytes and stromal fibroblasts may be sources of 
paracrine growth factors for EGF-R and erbB-3 products, respectively, that 
are expressed by the other cell types residing in close proximity in 
epidermal tissues. 
Given that both erbB and erbB-2 have been causally 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 malignancies. 
EXAMPLE 1 
Identification of a human DNA fragment related to the erbB proto-oncogene 
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., Rudikoff, S., & Aaronson, S. A., 1989, Proc. 
Nat. Acad. Sci. U.S.A. 86:802-806) or mammary tumor cell lines SK-BR-3, 
MDA-MB468, MDA-MB453, and MDA-MB415 (Kraus, M. H., Popescu, N. C., 
Amsbaugh, S. C. & King, C. R., 1987, EMBO. J. 6:605-610) have been 
described. For nucleic acid RNA hybridization, DNA and RNA were 
transferred to nitrocellulose membranes as previously described (Kraus, M. 
H., et al., 1987, supra). High stringency hybridization was conducted in 
50% formamide and 5xSSC at 42.degree. C. Filters were washed at 50.degree. 
C. in 0.1 xSSC. 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., Fanshier, L., 
Moscovici, C. & Bishop, J. M., 1980, J. ViroI. 36:575-585). 
Under reduced stringency hybridization, four SacI restriction fragments 
were detected. Two were identified as EGF-R gene fragments by their 
amplification 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.degree. 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 
Cloning 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 .lambda.sep6-lac5 was obtained from L. 
Prestidge and D. Hogness (Stanford University); many other cloning vectors 
derived from phage .lambda. or other genomes can be used for cloning this 
DNA fragment according to standard recombinant DNA methods that are well 
known in the art. Purified phage DNA was subjected to cos-end ligation, 
restriction with SacI, and fractionation in a continuous 10-40% sucrose 
gradient. A genomic library was prepared by ligating SacI 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 
representative clone with a 9 kbp 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 erbB was determined for both DNA strands by the 
dideoxy chain termination method (Sanger, F., Nicklen, S. & Coulson, A. 
R., 1977, Proc. Nat. Acad. Sci. U.S.A. 74:5463-67) using supercoiled 
plasmid DNA as template. The nucleotide sequence 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 Scientific Computing Laboratory. Hydrophobic and hydrophilic 
regions in the predicted protein were identified according to Kyte and 
Doolittle (Kyte, J. & Doolittle, R. F., 1982, J. MoI. BioI. 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. 2). 
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 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 tyrosine kinases was significantly lower, ranging 
from 39% to 46%. 
A single 6.2 kb specific mRNA was identified by Northern blot analysis of 
human epithelial cells using the 150 bp SpeI-AccI exon-containing fragment 
as probe (FIG. 2). Under the stringent hybridization conditions employed, 
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 
Cloning and characterization of cDNAs for the mRNA of the human erbB-3 
gene. 
In an effort to characterize the entire erbB-3 coding sequence, overlapping 
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 oligo dT-primed human placenta cDNA 
library in .lambda.gtll was obtained from Clontech. MCF-7 cDNA was 
prepared by first strand synthesis from 5 .mu.g poly A.sup.+ RNA using an 
oligo dT containing linker-primer and Mo-MuLV reverse transcriptase, 
followed by second strand synthesis with DNA polymerase I, RNaseH, and 
subsequent T4 DNA polymerase treatment. Double-stranded cDNA was 
directionally cloned into the SfiI site of .lambda.pCEV9 using specific 
linker-adapter oligonucleotides (Miki, T., Matsui, T., Heidaran, M. A. & 
Aaronson, S. A., 1989, Gene 83:137-146; see also, U.S. Application Ser. 
No. 07/386,053 of Miki et al., filed Jul. 28, 1989). 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, pE3-8, pE3-9, and pE3-11 carrying inserts with molecular 
weights ranging from 1.3 kbp to 4.3 kbp were isolated from a human 
placenta library, whereas the pE3-16 clone containing 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 contained 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 all 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 amino-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 extracellular 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 predicted erbB-2 and EGF-R protein, 
respectively. Within the extracellular domain, all 50 cysteine residues of 
the processed erbB-3 polypeptide were conserved and similarly spaced when 
compared to the EGF-R and erbB-2. Forty-seven cysteine residues were 
organized in two clusters containing 22 and 25 cysteines respectively, a 
structural hallmark of this tyrosine kinase receptor subfamily (see, for 
example, Yamamoto, T., Ikawa, 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 proteins (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 coincides with the minimal catalytic domain 
of tyrosine kinases (Hanks, S. K., Quinn, A. M. & Hunter, T., 1988, 
Science 241:42-52). There was significantly lower homology with other 
tyrosine kinases (FIG. 5). The consensus sequence for an ATP-binding site 
GxGxxG (Hanks, S. K. et al., 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 was 
its carboxyl terminus comprising 364 amino acids. This region showed a 
high degree of hydrophilicity and the frequent 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 positions 1260-1264 and was at both 
locations surrounded by charged residues, providing an environment of high 
local hydrophilicity. These observations render tyrosines 1197, 1199 and 
1262 likely candidates for autophosphorylation sites of the erbB-3 
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 .sup.3 H-label ed 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 gli are located in close proximity to 
erbB-3. 
EXAMPLE 5 
ErbB-3 expression in normal and malignant 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 observations indicate the preferential 
expression of erbB-3 in epithelial tissues and brain. 
ErbB-3 expression was also investigated in individual cell populations 
derived from normal human epithelial tissues including keratinocytes, 
glandular epithelial cells, melanocytes, and fibroblasts. For comparison 
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 
TABLE 1 
______________________________________ 
Normal expression pattern of human erbB 
gene family members. 
Relative 
Cell Source of Transcripts 
Gene RNA levels 
______________________________________ 
Embryonic fibroblast (M426) 
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 (NHEK) 
erbB-3 + 
erbB-2 + 
EGF-R ++ 
Glandular epithelium (AB589) 
erbB-3 (+) 
erbB-2 (+) 
EGF-R (+) 
Melanocyte (NHEM) erbB-3 ++ 
erbB-2 ++ 
EGF-R - 
______________________________________ 
Replicate Northern blots were hybridized with equal amounts (in cpm) of 
probes of similar specific activities for erbB3, erbB2, and EGFR, 
respectively. Relative signal intensities were estimated: - not 
detectable, (+) weakly positive, + positive, ++ strongly positive. 
derived from glandular epithelium. These findings are consistent with 
growth regulatory roles of all three receptor-like molecules 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 patterns of these 
receptor-like molecules were different in specialized cell populations 
derived from epidermal tissues. 
To search for evidence of erbB-3 involvement in the neoplastic process, 
erbB-3 mRNA levels in a series of human tumor cell lines were surveyed. 
The erbB-3 transcript was detected in 36 of 38 carcinomas and 2 of 12 
sarcomas while 7 tumor cell lines of hematopoetic origin lacked 
measureable 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 
rearrangement nor amplification was detected in the cell lines. FIG. 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 MDA-MB453 
(lane 3). Hybridization of the same filter with a human .beta.-actin probe 
(FIG. 7B) verified actual levels of mRNA in each lane. Densitometric 
scanning indicated that the erbB-3 transcript in each tumor cell line was 
elevated more than 100 fold above that of the control cell line. Thus, 
overexpression of this new member of the erbB family, as in the case of 
the EGF-R and erbB-2 genes, is likely to play an important role in some 
human malignancies. 
For purposes of completing the background description and present 
disclosure, each of the published articles, patents and patent 
applications heretofore identified in this specification are hereby 
incorporated by reference into the specification. 
The foregoing invention has been described in some detail for purposes of 
clarity and understanding. It will also be obvious that various changes 
and combinations in form and detail can be made without departing from the 
scope of the invention.