Patent Abstract:
observations regarding the role of muc1 in promoting the nuclear accumulation of egfr led us to propose the development of peptides to block nuclear accumulation of egfr as a means to block breast cancer progression . one exemplary peptide , the enls1 peptide , promotes cell death in breast cancer cell lines . studies in the mmtv - pymt mouse model of breast cancer demonstrate significant anti - tumor activity .

Detailed Description:
the inventors have discovered that egfr - related peptides , designed based on the egfr juxtamembrane region , can be used to block the nuclear translocation of egfr . the peptides include the tri - partite nuclear localization sequence of egfr , which can act in a dominant - negative fashion to block nuclear translocation of egfr . an egfr peptide is fused to or synthesized in tandem with a protein transduction domain ( e . g ., ptd - 4 ) to permit cellular uptake and intracellular action . one such peptide ( dubbed enls1 ) has been tested for its ability to block breast cancer cell growth and tumor growth in a transgenic mouse model , mmtv - pymt . other peptides also have been tested and found to have cell growth inhibitory properties . the egfr portion of the peptides can be based on the following sequence from egfr : lllvvalgiglfm rrr hiv rkr tl rr llqerelvepltps ( seq id no : 4 ). this sequence is juxtamembrane in the cytoplasmic domain of egfr . the tri - partite sequence ( underlined ) interacts with importin - 1β and is required for egfr nuclear localization ( hsu and hung , 2007 ; lo et al ., 2006 ). a portion of this sequence is linked to a protein transduction domain ( such as ptd4 previously described by bitler et al ., 2009 ). the linkage can be direct or through a linker . linkers can be , for example , runs of a single amino acid , or may be alternating runs of two amino acids . linkers can be , for example , from 1 to 25 residues in length . one polypeptide , dubbed enls1 , has the following sequence : yaraaarqara fmrrrhivrkrtlrrllqere ( seq id no : 8 ); the underlined sequence is ptd4 domain ). this 32 - mer peptide was made by synthesis ; it is highly water soluble and stable for & gt ; 1 week at 4 ° c . it can also be made by other techniques , including in recombinant cells . recombinant cells can be formed , for example , by transforming or transfecting cells with a recombinant vector which encodes for a polypeptide as described , operably linked to a transcription initiation site . the polypeptides of the invention do not occur in nature , but rather are made to juxtapose , a protein transduction domain with a sequence for blocking nuclear translocation of egfr . the two sequences can be made to immediately abut one another or can be separated by a linker . the blocking sequence does not comprise all of egfr , but instead comprises a subset of the egfr amino acid sequence . typically the size of the egfr sequence will be less than 50 residues , less than 40 , residues , less than 30 residues , or less than 25 residues . smaller sequences may also be used , provided that they inhibit the nuclear translocation of endogenous egfr . the nuclear translocation sequence may require one , two , or three of the arginine rich motifs , rrr , rkr , or rr , with or without the adjacent sequences in egfr . typically , the nuclear translocation sequence will be at least 6 , at least 8 , at least 10 , at least 12 , at least 14 , at least 16 , at least 18 , at least 20 residues in length . interestingly , permutations of the egfr amino acid sequence also have inhibitory properties , thus the sequence of the residues is not an essential feature of the cancer cell inhibitory or apoptotic activity . the protein transduction domain that is fused or linked to the egfr portion may comprise any protein transduction domain that is known in the art . these include without limitation ptd - 4 , hsv type i protein vp22 , and antenapedia protein transduction domain ( antp ). protein transduction domains enhance cellular entry of macromolecules . the domains may be natural or synthetic . spacers for use in fusion proteins may be additional amino acid residues that are used in fusion proteins typically to facilitate manufacture or synthesis . these can be fairly innocuous and typically are of a length of from 1 to 5 residues . the linkers can be monotonous or mixed residue . the residues can be random or sequences obtained from other proteins or designed for a particular property , e . g ., physical , chemical or biological . in one aspect , spacers provide an optimum distance between the two portions of the fusion protein . the polypeptides may be formulated in any suitable vehicle for administration . aqueous solutions may be used , but other alternatives may also be convenient , including capsules , time - release , implants , impregnated polymers , hydrophobic preparations , solids , powders , etc . the polypeptides can be combined with other anti - tumor agents , particularly with egfr inhibitors . the polypeptides can be administered by any means known in the art , including but not limited to intravenous injection , intramuscular injection , intratumoral injection , oral , subcutaneous injection , and intralymphatic . cancers and cancer cells which may be treated include breast , skin , colon , ovarian , prostate , cervical , colorectal , lung , brain , head and neck , pancreatic , kidney , and liver . one effect which may be observed upon administration is a reduced extent or retarded rate of initiation , growth , invasion , and metastasis . suitable assays for measuring these processes are described in the examples . other assays as are known in the art can be used as well . a person who is identified as having inherited genes associated with cancer is at increased risk of developing cancer . such persons can be treated to reduce their risk of cancer initiation . similarly , those who have been exposed to environmental risks , such as atomic bomb fall - out , nuclear fuel waste , and other pollutants , are at increased risk of developing cancer . genes which may be mutated and the autosomal dominant disorders they cause include , but are not limited to brca1 : breast cancer , brca2 : breast cancer , apc : colon cancer hnpcc : colon cancer , cdkn2 : melanoma . other autosomal dominant inherited cancer risks include basal cell nevus syndrome , neurofibromatosis type 2 , carney syndrome , osteochondromatosis , multiple , chordoma , familial , paraganglioma , familial , cowden syndrome , peutz - jeghers syndrome , esophageal cancer with tylosis , prostate cancer , gastric cancer , familial , renal cancer , familial , li - fraumeni syndrome , retinoblastoma , multiple endocrine neoplasia type 1 , tuberous sclerosis , multiple endocrine neoplasia type 2 , von hippel - lindau disease , neurofibromatosis type 1 , and wilms &# 39 ; tumor . autosomal recessive disorders disposing to cancer include ataxia - telangiectasia rothmund - thomson syndrome , bloom syndrome xeroderma pigmentosa , werner &# 39 ; s syndrome , and fanconi &# 39 ; s anemia . egfr inhibitors may be administered in the same treatment or prophylactic regimen as the peptides of the invention . they may be administered mixed with the peptides , or may be administered within a short time period of each other , such as within a week , within a day , or within a few hours or minutes . thus combinations of the inhibitors and the peptides may be formed ex vivo during manufacturing and formulation or in vivo subsequent to administration to a mammal . the two agents may be combined in a package , i . e ., a divided container to form a kit . any egfr inhibitor may be used , including kinase inhibitors . exemplary but not exhaustive , the list of inhibitors may include panitumumab , cetuximab , gefitinib , and erlotinib . the polypeptides may be useful in treatment , prevention , or adjuvant therapies . these words do not imply any particular level of cure or efficacy . rather , any time that a polypeptide is applied to a cell , tissue , or mammal so that a biological effect can be observed , a treatment has occurred . since no treatment is successful on all members of a particular disease population , it is only necessary that the treatment have an effect on a useful percentage of the population . prescreening and assessments may be performed to ascertain those individuals who will be most likely to benefit from the polypeptides of the invention . these will include those with a tumor such as a breast , brain , ovarian , colon , or pancreatic tumor . these tumors often contain altered egfr expression . other disease or predispositions can be assessed by determining the presence of an egfr mutation in a germline or somatic tissue of an individual . alternatively a biopsy tissue may be tested to determine the efficacy or likely efficacy of the polypeptide for that particular individual . family history of tumors may also be used as an indication that treatment may be beneficial . exposure to carcinogens may be used as an indication that treatment may be beneficial . animal models of various cancers and other diseases may be used to assess the effect of the polypeptides with other agents . combined additive or combined synergistic effects may be determined in animal models . cellular models and tissue models may also be used , typically in advance of animal models or clinical testing . the above disclosure generally describes the present invention . all references disclosed herein are expressly incorporated by reference . a more complete understanding can be obtained by reference to the following specific examples which are provided herein for purposes of illustration only , and are not intended to limit the scope of the invention . we first evaluated the ability of enls1 to inhibit the growth of breast cancer cell lines in culture . for these studies , we evaluated the effects of enls1 on the growth of mda - mb - 231 , and bt20 , both of which are positive for egfr expression and have a basal - like phenotype . we found enls1 to have not only a strong anti - growth phenotype , but also a very potent killing phenotype . treatment of triple - negative breast cancer cells ( both bt20 and mda - mb - 231 ) results in an induction of cell death as measured by mtt assay ( fig1 a ). we have also determined that enls1 can block tumor growth in the mmtv - pymt transgenic mouse model of breast cancer . we chose this model as it has been previously demonstrated to be an excellent model for human breast cancer ( lin et al ., 2003 ). furthermore , its initiation is not dependent on egfr activity , and is therefore an excellent model of spontaneous disease ( andrechek and muller , 2000 ). we began treatment of tumor bearing mmtv - pymt mice with 25 ug / g body weight enls1 , but this treatment was met with pulmonary toxicity ( data not shown ). we next began treatment of tumor bearing mmtv - pymt mice with 1 ug / g body weight enls1 , and this treatment gave no observable toxicities . based on this lack of toxicity , we began increasing the dosage of enls1 to 5 ug / g body weight , followed by 10 and 15 ug / g body weight . we did not see any apparent toxicity at any of these doses . we observed significant anti - tumor effects at both 10 and 15 ug / g body weight dosages , compared to either the lower concentration doses or a ptd4 control injection ( fig2 ). in both of these cases , we observed no significant growth of tumors in these mice , evaluating growth of tumors in 10 glands in each case . these results indicate that enls1 has potent anti - tumor activity in the mmtv - pymt mouse model of breast cancer . we have evaluated the efficacy of 14 different drugs for their ability to inhibit the growth of triple negative breast cancers . we have identified one of these drugs ( enls1 ), containing hegfr 622 - 42 , that effectively blocks tumor cell growth in both in vitro cell growth assays and in an established mouse model of breast cancer . we investigated the mechanism of action for this drug and found that it induces apoptosis , accompanied by membrane blebbing and reduction of nuclear localization of erbb1 . sequence identity indicates a block of erbb1 , erbb2 and erbb3 nuclear translocation . recent data from our lab has demonstrated a prominent role for muc 1 in promoting the nuclear activity of erbb receptors in driving transformation ( bitler et al ., journal of cell science , 2010 ). given this activity , we focused on blocking nuclear localization of erbb receptors and muc1 for developing anti - tumor peptides . the erbb receptors have significant homology in the nuclear localization sequence , all bearing a tri - partite basic amino acid sequence in their juxtamembrane region . in addition , a 3 residue basic sequence in the juxtamembrane of muc1 has also been shown to drive nuclear localization of muc1 . we therefore designed peptides around this region of egfr , and those sequences are as shown in table 1 . we next evaluated the ability of each of these peptides to inhibit the growth of the triple - negative breast cancer line , mda - mb - 468 . in our previous studies , we had demonstrated that these cells were particularly sensitive to a loss of muc1 expression for nuclear - egfr - dependent effects , including cyclin d1 expression . cells were evaluated for effects of peptide on growth inhibition by mtt assay at 1 , 3 and 5 days of 20 um peptide treatment . peptide concentration is based on evaluations of the ability of the peptide to block cell growth at 0 . 1 um - 100 um concentrations , where 20 um was found to optimally reduce cell growth in the active peptide , without any effects in the control peptide ( data not shown ). we then treated mda - mb - 468 cells with 14 derivatives of the peptide , including versions with the full sequence , portions of the sequence , and acidic substitutions for the basic amino acids ( which were synthesized as controls ) ( table 1 ). we found that a key 21 amino acid sequence is optimal for causing cell death , which is encompassed by enls1 ( fig1 ). in addition , while data shown is for mda - mb - 468 cells , growth inhibitory effects were observed for mda - mb - 231 and bt20 cells as well ( both triple negative breast cancers ). we then proceeded to evaluate cell death in other cell types , including her2 and her3 positive cells , and her negative cells . in addition , we evaluated the efficacy of enls1 on pancreatic cells . surprisingly , we found that enls1 effectively blocks cell growth in 2 different pancreatic cell lines ( bxpc3 and aspc1 ) as well as her2 positive t47d breast cancer cells . it had no effect , though , on hek293 cells , which do not express the her receptors ( data not shown ). we next set out to determine whether cell death was occurring by the induction of apoptosis . for this , we treated cells for either 30 minutes or 2 hours and made protein lysates ( fig2 a ). lysates were then analyzed by sds - page and immunoblot for expression of cleaved caspase 3 and cleaved parp , both markers of the induction of apoptosis . we found that by 2 hours of peptide treatment , both were induced , indicating that cells were dying by apoptosis . in addition , we found that these cancer cells had established a state of autophagy for survival ( as demonstrated by expression of atg12 ). enls1 was also observed to block the formation of atg12 , indicating a loss of the cell preserving the autophagic state . no effects on protein expression for any proteins evaluated were affected by our control peptide . in addition , we observed a loss of cyclin d1 expression , which is known to be induced by nuclear egfr . finally , we found a loss of pakt expression , which we subsequently found to correlate with a loss of total akt expression . we are currently evaluating the mechanism by which this may be occurring . we next set out to verify that we are blocking the nuclear translocation of egfr , which we evaluated by fractionating the cytoplasmic , membrane and nuclear fraction of cells treated with enls1 versus enlscp5 ( the control peptide with 3 key amino acids changed from basic ( arginine ) to acidic ( aspartic acid ). in fig2 b , we demonstrate that while egfr is phosphorylated on residue 845 ( p - egfr ) in response to egf treatment , this form of egfr does not translocate to the nucleus in the presence of enls1 , whereas it readily translocates to the nucleus in the control ( either vehicle or cp4 control peptide ). enls4 also inhibits the nuclear translocation of p - egfr , as enls4 is the minimal nuclear translocation sequence of egfr . interestingly , enls4 has minimal effects on cell growth ( fig1 ), indicating that the loss of egfr translocation can inhibit cell growth , but that another function must be being affected by enls1 . to determine how enls1 was driving the cells to undergo apoptosis , we began by evaluating the morphological changes that are observed upon enls1 treatment . we noticed that within a few hours of peptide treatment , cells began to exhibit membrane blebbing , demonstrating a membrane ‘ bubbling ’ ( data not shown ). we decided to look by electron microscopy to determine what changes were occurring at a sub - structural level . upon performance of scanning electron microscopy , we were able to identify the formation of multiple vesicles under the plasma membrane of enls1 treated cells , but not in control - treated cells ( fig3 ). we are currently investigating the origin of these vesicles through investigations into membrane integrity , calcium flux and exocytosis in the cell . enls1 inhibits tumor growth and metastasis in the mmtv - pymt mouse model of breast cancer we have been extensively testing the ability of enls1 to affect breast cancer growth in vivo . we have been testing the mmtv - pymt model of breast cancer , a model that forms spontaneous breast tumors in all 10 mammary glands of the mouse . this model has been extensively evaluated by pathologists , and shown to undergo many of the same changes that occur in human breast cancer . in addition this is a metastatic model , with metastases forming in the lung in close to 100 % of the animals . mmtv - pymt mice were allowed to develop tumors of at least 0 . 5 cm in diameter prior to treatment , and were then treated for 15 days with either 1 ug / g body weight ( n = 1 ), 5 ug / g body weight ( n = 1 ), 10 ug / g body weight ( n = 4 ), 15 ug / g body weight ( n = 1 ), or 40 ug / g body weight ( n = 1 ). this was done initially to determine if treatment with enls1 would result in toxicity . we found that no toxicity to the animal ( as determined by evaluated changes in weight , grooming , gait ) was observed at any of the treatment groups . we next began to evaluate the effects of peptide treatment on tumor growth . the mmtv - pymt model is a stochastically developing tumor model , with tumors developing in all ten mammary glands from as early as 6 weeks of age . this allows us to determine both how drug treatment effects established tumors as well as newly initiated tumors . fig4 a shows that enls1 exhibits strong antitumor activity , as shown by the tumor growth rate in the animals treated , also that this effect on tumor growth increases as the amount of drug increases . in fig4 b , we demonstrate that enls1 treatment inhibits the initiation of new tumors during the treatment . finally , we found a striking reduction in lung metastases in treated mice versus control mice . this model is known to be 100 % metastatic to the lungs , and we therefore excised the lungs of untreated and control mice to evaluate effects of enls1 on lung metastasis . we found only 2 / 9 mice that had been treated with enls1 with visible lung metastases , while all of the control mice ( 5 / 5 ) had lung metastases . inhibition of tumor growth in the mmtv - pymt model is associated with apoptosis finally , we have evaluated the molecular events occurring in the tumors excised from treated mice . mice that had received 10 ug / g body weight of enls1 for 15 days were sacrificed and protein lysates prepared from the remaining tumors . we then evaluated these protein lysates for expression of cleaved parp as an indicator of the induction of apoptosis , and found a significant increase in cleaved parp in enls1 treated tumors ( fig5 ). we also evaluated tumors for the expression of cyclin d1 , py845egfr , and muc1 , but did not observe any significant differences . anders , c ., and carey , l . a . 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