Patent Application: US-15935302-A

Abstract:
the present invention discloses a system and method using the human erbb3 receptor , p85 - serbb3 , as a negative regulator of heregulin - stimulated erbb2 , erbb3 , and erbb4 activation . the present invention also discloses a system and method of p85 - serbb3 binding to heregulin with an affinity comparable to that of full - length erbb3 , and competitively inhibiting high affinity heregulin binding to erbb2 / 3 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 and uses p85 - serbb3 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 preparing the protein . the present invention discloses a system and method of therapeutic applications in human malignancies associated with heregulin - mediated cell growth such as breast and prostate cancer .

Description:
using various recombinant soluble 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 / erbb 1 inhibits tgfα a dependent transformation . soluble egfr secreted by the a43 1 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 egf / erbb 1 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 the physiological role of p85 - serbb3 in normal tissues also has not been understood . the present invention indicates that 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 enough 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 an 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 . evidence suggests 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 for 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 : 4 and seq id no : 2 . the present invention also defines the biochemical and biological characteristics of a novel serbb3 isoform designated p85 - serbb3 . the present invention discloses 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 . this novel erbb3 receptor isoform , therefore , is disclosed as a potent modulator of hrg regulated cell proliferation and differentiation in normal human tissues , and 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 hrgβ egf domain 177 - 241 ( hrgα ) and hrgβ1 1176 - 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 hrga , 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 r31 f encoding p85 - serbb3 ( ref ) 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 - hc1 , ph 7 . 5 , 150 mm nac1 , mm mnc1 2 , and 1 mm cac 1 2 ) and eluted using column buffer containing 1 m αmethyl d - mannoside , then dialyzed against 20 mm tris - hc1 , 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 nac1gradient containing 20 mm tris - hc1 , 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 erbb3 . 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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( i ) sequence characteristics : ( ii ) length ( iii ) type ( iv ) molecule type ( v ) original source : ( vi ) organism : homo sapience ( i ) sequence characteristics : ( ii ) length ( iii ) type ( iv ) molecule type ( v ) original source : ( vi ) organism : homo sapience ( i ) sequence characteristics : ( ii ) length ( iii ) type ( iv ) molecule type ( v ) original source : ( vi ) organism : homo sapience 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 .