Patent Application: US-1861008-A

Abstract:
the present invention discloses a method using human soluble erbb3 , for example p85 - serbb3 , as a negative regulator of heregulin - stimulated erbb2 , erbb3 , and erbb4 activation . the present invention also discloses p85 - serbb3 binding to heregulin with an affinity comparable to that of full - length erbb3 , and competitively inhibiting high affinity heregulin binding to erbb2 / erbb3 heterodimers on the cell surface of breast carcinoma cells . the present invention also uses p85 - serbb3 to inhibit heregulin - induced phosphorylation of erbb2 , erbb3 , and erbb4 in cells , as a negative regulator of heregulin - stimulated signal transduction , and as a block for cell growth . the present invention is also directed to nucleic acids and expression vectors encoding p85 - serbb3 , host cells harboring such expression vectors , and methods of producing the protein . the present invention discloses a method of therapeutically treating human malignancies associated with heregulin - mediated cell growth such as breast and prostate cancer .

Description:
using various recombinant forms of egfr , it has been shown that efficient inhibition of full - length egfr activation by dominant - negative heterodimerization occurs only when these deletion mutants retain the transmembrane domain and the extracellular domain . similarly , a recombinant dominant - negative erbb3 mutant with a deleted cytoplasmic domain but which retains its transmembrane domain can inhibit full - length erbb2 and erbb3 activation . in contrast , in avian tissues , expression of a naturally occurring segfr / erbb1 inhibits tgfα dependent transformation . an aberrant soluble egfr secreted by the a431 human carcinoma cell line also has been reported to inhibit the kinase activity of purified full - length egfr in a ligand - independent manner . these soluble egfr / erbb1 receptors do not function as antagonists through high affinity ligand - binding . similarly , herstatin , a naturally occurring soluble erbb2 protein which inhibits erbb2 activation appears to function by blocking erbb2 dimerization ; this inhibition is thought to be mediated via ligand - independent binding of herstatin to erbb2 . in contrast , serbb3 , including p85 - serbb3 and p45 - serbb3 , inhibits hrg - induced stimulation of erbb2 , erbb3 , and erbb4 , at least in part , by neutralizing ligand activity through competitive binding . the present invention discloses that p85 - serbb3 is capable of binding to the cell surface . the physiological role of p85 - serbb3 in normal tissues also has not been understood to date . as discussed in greater detail below , although a much higher concentration ( 100 - fold ) was required to inhibit cell growth , a 10 - fold molar excess of p85 - serbb3 was sufficient for inhibition of phosphorylation of erbb receptors . at this ratio , a small fraction of receptors are still activated and are sufficient for growth stimulation . it is known that the 2 . 1 kb transcript encoding p85 - serbb3 is expressed at low levels compared to the full - length c - erbb3 transcript in all cell lines and tissues examined to date , however , local expression of this transcript has been studied in detail . it is , therefore , plausible that p85 - serbb3 acts as a hrg antagonist locally in a tissue - specific and / or stage - specific manner , and related studies to examine the distribution of p85 - serbb3 in selected tissues are currently underway . research in the field shows that local concentrations of autocrine growth factors such as egf are exquisitely regulated and do not travel far from the cell surface from which they are released . in this context , tightly regulated levels of local p85 - serbb3 expression have important consequences on cellular activities , such as hrg - mediated cell growth . these consequences are even more dramatic in cancer cells where cell polarity is typically lost , resulting in deregulation of normal spatial and temporal control of growth factor : receptor interactions . the present invention provides several novel isolated and purified nucleic acids which encode isoforms of erbb3 and nucleic acids encoding engineered variants of these proteins . preferred embodiments are nucleic acids which specifically encode isoforms of erbb3 whose amino acid sequence comprises the sequence of seq id no : 2 and seq id no : 4 . the present invention also defines the biochemical and biological characteristics of a novel serbb3 isoform designated p85 - serbb3 . embodiments of the present invention relate to the use of p85 - serbb3 as a unique hrg inhibitor because it can block hrg binding to cell surface receptors via binding to hrg with high affinity , thereby , inhibiting hrg - induced stimulation of erbb2 , erbb3 , and erbb4 . this inhibition is sufficient to effectively block hrg - stimulated cell growth . these novel serbb3 receptor isoforms , therefore , are potent modulators of hrg regulated cell proliferation and differentiation in normal human tissues , and as such provide an ideal candidate for the development of novel cancer therapeutics . conditioned media from cells expressing p45 - serbb3 and p85 - serbb3 inhibit hrg activation of erbb3 . p45 - serbb3 and p85 - serbb3 are naturally occurring secreted products of the erbb3 gene ( lee and maihle 1998 ). p45 - serbb3 contains the amino - terminal 310 amino acids of erbb3 and two unique carboxy - terminal amino acid residues . p85 - serbb3 contains the amino - terminal 519 amino acids of erbb3 and 24 unique carboxy - terminal amino acid residues ( see fig1 ). to examine whether p45 - serbb3 and p85 - serbb3 can modulate hrg receptor activation cells stably transfected with these corresponding cdna clones were isolated . these cells secrete p45 - serbb3 and p85 - serbb3 into the culture medium ( see fig2 a ). the conditioned medium from these cells was used as the source of p45 - serbb3 or p85 - serbb3 in a series of preliminary experiments described below . to test the ability of p45 - serbb3 and p85 - serbb3 to modulate aspects of hrg - mediated erbb receptor activation a clonal derivative of the ba / f3 cell line expressing exogenous erbb2 and erbb3 was stimulated with hrgα egf domain 177 - 241 ( hrgα ) and hrgβ1 176 - 246 ( hrgβ ) in the absence or presence of concentrated conditioned media containing p45 - serbb3 and p85 - serbb3 . as shown in fig2 , hrgβ was at least 20 - fold more effective than hrgα in stimulating erbb3 tyrosine phosphorylation . conditioned media containing serbb3 inhibited hrgα - stimulated erbb3 activation by 40 % ( p45 - serbb3 ) and 80 % ( p85 - serbb3 ) at 1 μg / ml hrgα , as determined by densitometric analysis . however , at a higher concentration ( 2 μg / ml ), conditioned media containing p85 - serbb3 decreased activation by 30 %, although inhibition by conditioned media containing p45 - serbb3 was negligible ( see fig2 a ). in the presence of conditioned medium containing either p45 - serbb3 or p85 - serbb3 , ligand stimulation of erbb3 tyrosine phosphorylation was decreased by 60 % and 90 %, respectively , at both 50 and 100 ng / ml hrgβ ( see fig2 c ). these data indicate that p85 - serbb3 inhibited erbb3 phosphorylation in response to both hrgα and hrgβ more effectively than p45 - serbb3 , although the concentration of p85 - serbb3 used in these studies was lower than that of p45 - serbb3 ( fig2 a ). purification of p85 - serbb3 . p85 - serbb3 was isolated from a concentrated conditioned medium of cells stably transfected with a cdna clone r31f encoding p85 - serbb3 and was purified in two steps . the first step was lectin affinity chromatography with a con a column ( sigma ). the bound p85 - serbb3 was washed with column buffer ( 10 mm tris - hcl , ph 7 . 5 , 150 mm nacl , 1 mm mncl 2 , and 1 mm cacl 2 ) and eluted using column buffer containing 1 m α - methyl d - mannoside , then dialyzed against 20 mm tris - hcl , ph 7 . 5 overnight . the second step of purification was accomplished using a mono q ®, an ion exchanger for resolution of proteins and peptides , ion exchange fplc ®, i . e ., a microprocessor controlled , solvent delivery apparatus used in purification of biologically active compounds column ( pharmacia ). the bound p85 - serbb3 was eluted from the column with 0 - 500 mm nacl gradient containing 20 mm tris - hcl , ph 7 . 5 . samples taken from each step were subjected to sds - page in duplicate and analyzed by coomassie staining and by western blot using anti - erbb3 236 antibody recognizing the extracellular domain of the erbb3 ( lee and maihle 1998 ). the final p85 - serbb3 pool was homogeneous on sds - page , and the identity of the purified protein was confirmed by western blot analysis . purified preparations of p85 - serbb3 were used in all subsequent experiments . p85 - serbb3 binds to hrg with high affinity . previous reports based the assignation of the subdomain boundaries of the erbb3 extracellular domain on the subdomain boundaries of egfr ( lee and maihle 1998 ) as defined by the genomic structure of avian erbb1 ( callaghan , antczak et al . 1993 ). accordingly , p85 - serbb3 is composed of subdomains i through iii and includes the first 45 amino acids of subdomain iv ( aa 1 - 519 ), and a unique twenty - four amino acid sequence at the carboxy - terminus . binding studies using heregulins indicate that subdomains i and ii are required for heregulin binding ( singer , landgraf et al . 2001 ). on the other hand , for egf binding to egfr subdomains i and iii are low and high affinity binding sites , respectively ( lax , bellot et al . 1989 ). because p85 - serbb3 contains both subdomains i through iii the present invention determined that p85 - serbb3 would bind to heregulins . direct binding between p85 - serbb3 and radiolabeled hrgβ was examined using the chemical crosslinker bs 3 . as shown in fig3 a , a protein complex of 90 kda was formed between p85 - serbb3 and 125 i - hrgβ . formation of this complex could be inhibited by addition of excess cold hrgβ but not by addition of excess insulin , indicating that p85 - serbb3 binding to hrgβ is specific and that purified preparations of p85 - serbb3 are biologically active . an analysis of 125 i - hrgβ 177 - 244 binding to immobilized p85 - serbb3 was then performed using an erbb3 - igg homodimer as a positive control . as shown in fig3 , p85 - serbb3 binds to hrgβ 177 - 244 with a k d of 3 . 0 ± 0 . 2 nm . in comparison , erbb3 - igg binds to hrgβ 177 - 244 with a k d of 4 . 7 ± 0 . 2 nm . these results demonstrate that p85 - serbb3 binds to hrgβ 177 - 244 with an affinity similar to that of the extracellular domain of erbb3 . based on the results of these two complementary experimental approaches , the present invention establishes the use of p85 - serbb3 to bind to hrg with an affinity equivalent to the affinity of hrg for the full - length extracellular domain of erb3 . p85 - serbb3 inhibits binding of hrg to receptors on the cell surface . the present invention also discloses the use of p85 - serbb3 to effectively limit binding of heregulin to cell surface receptors in the breast carcinoma cell line t47d . this cell line expresses all four erbb receptors at moderate levels . cells were incubated with varying concentrations of p85 - serbb3 in the presence of 125 i - labeled hrgβ 177 - 244 . simultaneously , a separate group of cells was incubated with 125 i - hrgβ 177 - 244 in the presence of varying concentrations of 2c4 , a monoclonal antibody specific for the erbb2 extracellular domain ( lewis , lofgren et al . 1996 ). as shown by the inhibition curves ( see fig4 ), p85 - serbb3 and 2c4 inhibit hrgβ 177 - 244 binding to cell surface receptors with similar ic 50 values ( 0 . 45 ± 0 . 03 nm and 0 . 55 ± 0 . 03 nm , respectively ) although the mechanism of inhibition by these two molecules is distinct . although 2c4 inhibits heregulin binding to cell surface receptors by blocking erbb2 - erbb3 heterodimerization via binding to the erbb2 extracellular domain ( fitzpatrick , pisacane et al . 1998 ), p85 - serbb3 inhibited receptor activation , at least in part , by competing for heregulin binding to the cell surface . p85 - serbb3 blocks hrg - induced activation of erbb2 , erbb3 , and erbb4 . the present invention also examined the ability of p85 - serbb3 to modulate hrg - stimulated receptor activation in the ba / f3 ( erbb2 + erbb3 ) cell line using purified p85 - serbb3 . this allowed an analysis of the mechanism of p85 - serbb3 mediated inhibition in a quantitative manner . as shown in fig5 , when ba / f3 ( erbb2 + erbb3 ) cells were treated with p85 - serbb3 at a 10 - fold molar excess over hrgβ 1 - 241 , erbb3 phosphorylation levels were reduced to basal levels . a similar level of receptor inhibition also was apparent when either a 2 . 5 - or 5 - fold molar excess of p85 - serbb3 was used in these experiments . exogenous addition of p85 - serbb3 also inhibited hrg - induced erbb2 activation . p85 - serbb3 blocked hrg stimulation whether the cells were treated with the egf - like domain of hrg ( hrgα or hrgβ ), as shown in fig2 , or with hrgβ 1 - 241 ( see fig5 ), suggesting that inhibition by p85 - serbb3 occurs , at least in part , through a direct interaction between p85 - serbb3 and the egf - like domain of hrg . cells treated with the same concentration of p85 - serbb3 but not stimulated with hrg did not exhibit altered erbb2 or erbb3 tyrosine phosphorylation , or show any change in the level of either erbb2 or erbb3 expression , suggesting that p85 - serbb3 does not function as a “ ligand ” for these receptors . to examine whether exogenous addition of p85 - serbb3 exerts the same inhibitory effect on endogenously expressed erbb receptors , and to determine whether p85 - serbb3 could modulate other members of the egf receptor family , the activity of p85 - serbb3 in two breast carcinoma cell lines , i . e ., t47d and mcf7 , was tested . as shown in fig6 a , addition of p85 - serbb3 ( at a 6 - fold molar excess relative to hrgβ ) inhibited hrg - induced activation of erbb2 , erbb3 , and erbb4 in both the t47d and mcf7 cell lines . in contrast , at least in these two cell lines which express low egfr levels , egfr phosphorylation remained at basal level in cells treated with hrgβ regardless of whether p85 - serbb3 was present or not . similarly , egf - induced phosphorylation of egfr or erbb2 , or , to a lesser degree , phosphorylation of erbb3 , was not decreased by p85 - serbb3 . these results demonstrate that inhibition by p85 - serbb3 is specific for hrg - induced activation of erbb2 , erbb3 , and erbb4 . it is notable that in the t47d cells , a decrease in erbb2 , erbb3 , and erbb4 protein levels following hrg stimulation was observed . in mcf7 cells a decrease in erbb3 levels also was apparent when hrg was added to the culture medium ( see fig6 a ). it has been reported that the polyclonal erbb3 antibody specific to the carboxy - terminal 17 aa used in this study preferentially recognizes non - phosphorylated erbb3 on western blots ( vartanian , goodearl et al . 1997 ). thus , when t47d or mcf7 cells are stimulated with hrg , a significant fraction of erbb3 is phosphorylated , and , therefore , undetectable with this particular erbb3 antibody . the anti - erbb antibodies used in these experiments recognize the carboxy - terminal 17 aa ( erbb3 ) and 18 aa ( erbb2 and erbb4 ) sequences of these receptors . each of these sequences contains one tyrosine residue . immunoblot detection by the anti - erbb2 and erbb4 antibodies used in this study , therefore , may reflect either the level of receptor expression or the unphosphorylated fraction of these receptors . p85 - serbb3 inhibits activation of downstream effectors of hrg . hrg - stimulated activation of erbb2 , erbb3 , and erbb4 leads to activation of two major signal transduction pathways : the pi3k pathway and the mapk pathway ( wallasch , weiss et al . 1995 ). the present invention tested whether p85 - serbb3 could inhibit activation of these two downstream effector pathways in t47d cells . specifically , the present invention examined activation of mapk and akt by analyzing the phosphorylation levels of these proteins , and examined the ability of p85 phosphatidylinositide 3 - kinase (“ pi3k ”) to interact with erbb3 following hrgβ treatment . in the presence of p85 - serbb3 ( 10 - fold molar excess relative to hrgβ ), tyrosine phosphorylation of erbb3 was reduced to basal levels . in the same cell population , addition of exogenous p85 - serbb3 abrogated the phosphorylation of both mapk and akt as determined by western blot analysis , and inhibited erbb3 &# 39 ; s association with p85 pi3k ( see fig6 b ). these results further demonstrate that p85 - serbb3 can inhibit the activation of erbb2 , erbb3 , and erbb4 , and this inhibition affects the activation of downstream signaling molecules such as mapk , akt , and pi3k . p85 - serbb3 inhibits hrg - stimulated cell growth . the present invention also discloses the inhibition of hrg - induced phosphorylation of erbb receptors by p85 - serbb3 as correlated with the modulation of hrg &# 39 ; s biological effects . specifically , a cell growth assay using mcf7 cells stimulated with hrgβ was performed and showed that , within the concentration range tested , growth of this cell line was dose - dependent ( see fig7 ). it was observed that at a concentration of 0 . 4 nm hrgβ the cell growth rate was half of the rate observed at saturating levels of hrgβ . in cell cultures grown in the presence of 0 . 4 nm hrgβ and p85 - serbb3 ( a 100 - fold molar excess relative to hrgβ ), p85 - serbb3 inhibited cell growth by 75 % and 90 %, at densities of 5 , 000 and 8 , 000 cells / well , respectively , whereas the same concentration of p85 - serbb3 did not affect cell growth in the absence of hrgβ ( see fig7 ). thus , the present invention discloses the use of p85 - serbb3 as a potent inhibitor of hrg - dependent breast carcinoma cell growth in vitro . alimandi , m ., m . heidaran , et al . 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