Patent Application: US-11380002-A

Abstract:
an isolated peptide or polypeptide containing the sequence vsiglsfpmlp , found in the secreted form of human muc1 , that enhances an immune response when administered to a mammal , compositions containing the peptide , host cells producing the peptide and methods of use . the peptide or polypeptide may be conjugated to a carrier protein and administered as part of a vaccine or immunogenic composition for prevention or treatment of a disease or disorder .

Description:
in order to study the immune responses against a mammary tumor using a defined tumor antigen , we transfected da - 3 cells , a mammary tumor cell line that leads to metastatic lesions and death of balb / c mice hosts , with the transmembrane muc1 isoform ( da - 3 / tm ), with the secretory form ( da - 3 / sec ), or with the neomycin vector alone ( da - 3 / neo ). transfection with the secretory form of muc1 renders the da - 3 cells incapable of growing in intact balb / c mice , while they grow in nude balb / c animals . vaccination with da - 3 / sec cells confers protection to challenge with the da - 3 / tm or da - 3 / neo cells and against two unrelated tumors also syngeneic to balb / c mice . it appears that a unique 11 amino acid peptide present in the secreted muc1 isoform is involved in the protective effect and serves as an immunoenhancer molecule . mice and tumors . intact balb / c mice were maintained by brother - sister matings in our laboratory at the university of miami school of medicine . balb / c nu +/ nu + were purchased from taconic labs . the da - 3 tumor cell line was derived from the in vivo d1 - dmba - 3 mammary tumor syngeneic to balb / c mice ( 4 ). the da - 3 cells produce tumors in balb / c mice and cause metastatic lesions in the lung . the cell line is grown in rpmi - 1640 media supplemented with 5 % fcs ( fetal calf serum ), 100 μpenicillin , 100 μg / ml streptomycin , 5 × 10 − 5 m 2 - beta - mercaptoethanol ( 2 - bme ), 2 mm l - glutamine , 1 % nonessential amino acids , 1 % essential amino acids , and 1 % sodium pyruvate ( all from gibco brl , gaithersburg , md . ), and maintained by serial passage . these cells are free of endotoxin as ascertained by routine assays with limulus amebocyte lysate ( pyrogent ® plus ; whittaker m . a . bioproducts , inc ., walkersville , md .). preparation of peptide fragments and antibodies . the monoclonal antibody designated h23 was used to detect the tandem repeat sequence common to both muc1 / tm and muc1 / sec . antibody 1709 ( ayes labs , tigard , oreg ) was prepared by immunizing chickens with the muc 1 / sec specific peptide vsiglsfmlp ( seq id no : 1 ) conjugated to klh with malaimide - activated keyhole limpet hemocyanin ( klh ). free peptide concentrations and conjugations of at least 150 peptide molecules per klh molecule were utilized for immunization . pre - immune igy antibody was prepared from eggs collected prior to immunization . transfection of da - 3 cells . stable transfectants expressing muc1 isoforms were generated by co - transfecting an expression plasmid harboring either muc1 / tm or the secreted form muc1 / sec cdna with the neomycin plasmid ( psv2 neo ) selection marker into da - 3 mouse mammary tumor cells . as control , cells were also transfected with the empty plasmid and selected for neomycin resistance . tumor growth in vivo . the parental da - 3 cells , the neomycin control and muc1 isoforms transfected cells were washed , counted , and 10 6 cells were injected subcutaneously ( s . c .) in syngeneic intact or nude ( nu + / nu + ) balb / c mice . tumor growth was monitored every 2 - 3 days and mice bearing a tumor of & gt ; 10 mm as measured with a caliper were scored as tumor positive . vaccination protocols . balb / c mice were vaccinated two or three times two weeks apart with 10 6 da - 3 / sec tumor cells in a physiological saline solution . two weeks after the last vaccine administration the animals were challenged with da - 3 cells untransfected or transfected with neomycin vector alone or with the muc1 transmembrane isoform . in some experiments the tumors used to challenge were the renal cell carcinoma , renca , or the osteosarcoma k7 . 10 6 tumor cells were used for each challenge mixed with 10 6 da - 3 / sec cells . in other studies the unique 11 amino acid peptide present in the secreted muc1 isoform was synthesized by aves labs , inc . ( tigard , oreg .) and bound to klh . because the exact ratio of peptide to klh varies with each synthesis ; the preparations were normalized to deliver the same amount of peptide in each administration . the klh peptide was used as a vaccine two weeks apart . fifty μg of peptide was given to balb / c mice with complete freund &# 39 ; s adjuvant for the first vaccination . two weeks later 25 μg of the peptide was administered with incomplete freund &# 39 ; s adjuvant . after two weeks the mice were challenged with 10 6 da - 3 / sec or da - 3 / neo cells mixed with 25 μg of peptide with no adjuvant . in all studies unvaccinated mice were given the same tumor challenge as in the experimental groups to serve as controls . tumor growth was monitored every two or three days and tumor size was measured by caliper . da - 3 mammary tumor cells were transfected as described in materials and methods with either the transmembrane and secretory isoforms of the human muc1 . the success of the transfections was proven by staining the cells for the presence of the muc1 tandem repeat using the h 23 antibody specific for this sequence ( 5 ). the resulting cell lines were used in in vivo experiments to determine the incidence and time of tumor appearance in balb / c mice . as seen in table 1 , implantation of da - 3 , da - 3 / neo and da - 3 / tm mammary tumor cells into mice gave rise to palpable tumors of approximately the same size by seven days and by 15 days essentially all animals had sizable tumors . surprisingly , the da - 3 cells transfected with the muc1 secreted form ( da - 3 / sec ) failed to cause tumor development . remarkably , as seen in fig1 , these animals remain tumor free even after more than 12 months after implantation , while animals with the other three types of da - 3 cells fail to survive past 100 days . it should be pointed out that no tumor growth was observed in mice that were implanted with the da - 3 / sec tumor cells up to a concentration of 1 × 10 7 cells per inoculation . a trivial explanation to these results could be that the transfection process had selectively impaired the basic growth potential of the da - 3 / sec cells . to test this possibility we investigated the in vitro growth characteristics of the four types of da - 3 tumor cells . as shown in fig2 , the parent cell line and all the various transfectants grew with similar kinetics in vitro . in fact , the da - 3 / sec cells seemed to proliferate better than the other cell lines , indicating that the in vitro growth potential of these cells has not been altered by the transfection manipulations . in order to determine whether the da - 3 / sec cells had lost all tumorigenic potential in vivo , all four da - 3 cell lines were implanted in nu + / nu + balb / c mice and the incidence of tumor appearance and tumor size at various times were assessed . fig3 shows that the da - 3 , da - 3 / neo , and da - 3 / tm cells cause palpable tumors by seven days after implantation in balb / c nude mice and they grew in a manner similar to that of intact balb / c animals . in contrast with the results in table 1 and fig1 , da - 3 / sec tumor cells gave rise to tumors in 30 % of all nude mice by 14 days and by 25 days all these animals had tumors . thus , the lack of tumor growth in the intact balb / c mice implanted with the da - 3 / sec cells appears to be immunologically controlled , since implantation of this tumor in nude mice resulted in 100 % tumor takes , albeit at a slower time of appearance . these results were repeated using other two da - 3 tumor cells separately transfected with the expression plasmid harboring the secreted form of muc1 . as seen in table 2 , none of the three separate da - 3 cells transfections with the muc1 - sec gene were capable of growing in intact balb / c mice . fig4 shows that the three separate da - 3 / sec cell transfectants , i . e . da - 3 / sec , da - 3 / sec 11 , and da - 3 / sec 22 , could all grow in vivo in balb / c nude mice , although their growth kinetics in the immunodeficient mice were somewhat different . interestingly , da - 3 / sec tumors that grew in nude balb / c animals did not grow when implanted in immunologically intact balb / c mice , but do grow when inoculated with other nude balb / c animals ( data not shown ). the results of these experiments suggest that t cells , and to a lesser degree another type of non t cell effector , are involved in the control of growth of tumors transfected with the secreted form of muc1 . we evaluated whether implantation of balb / c mice with da - 3 / sec cells could confer protection to other muc1 expressing tumor cells . da - 3 cells expressing no mucin ( da - 3 / neo ) were also included in the study . experimental groups received two or three injections one week apart of 1 × 10 6 da - 3 / sec cells prior to challenge with 1 × 10 6 cells da - 3 / tm or da - 3 / neo cells alone or mixed with da - 3 / sec cells . as controls , unvaccinated animals were challenged with either da - 3 / neo or da - 3 / tm cells . animals receiving two injections of unmixed da - 3 sec ( 10 6 cells ) caused only a short delay in the appearance of tumors when challenged with da - 3 / tm cells alone , i . e . 13 / 22 animals with tumors at two weeks as compared to 13 / 14 animals with tumors at one week in the control group . mice that had received three injections of 10 6 da - 3 / sec cells before challenge unexpectedly retarded the growth of not only da - 3 / tm cells but also da - 3 / neo cells compared to the unvaccinated control groups . remarkably , when the da - 3 / tm tumor cells used for challenge were mixed with the da - 3 / sec cells at the time of implantation ( fig5 ), there was not only a retardation in the time of tumor appearance compared to the control groups , but there was an actual substantial protection resulting in lack of growth of the da - 3 / tm tumor cells in 50 % of the vaccinated mice . furthermore , this protection did not appear to be due to a recognition of the muc1 molecule , since a similar effect could be seen in animals vaccinated with da - 3 / sec cells and challenged with a mixture of da - 3 / neo and da - 3 / sec mammary tumor cells . further studies were carried out to determine whether vaccination with da - 3 / sec cells could confer protection against the growth of tumors other than those in da - 3 mammary tumor background . two unrelated tumors syngeneic to balb / c mice were employed , i . e . a renal cell carcinoma line , renca , and an osteosarcoma , the k7 cell line . fig7 shows that vaccination of balb / c mice twice with the da - 3 / sec cells followed by a challenge with a mixture of renca cells and da - 3 / sec cells resulted in substantial reduction of tumor growth as compared to the unvaccinated controls . the addition of the da - 3 / sec at the time of challenge was necessary , as was the case with the da - 3 / tm and da - 3 / neo cell experiments , since the unmixed renca cells growth was not impaired . similar results were obtained in experiments where the k7 osteosarcoma cells were used . as seen in fig8 , 80 % of unvaccinated mice implanted with a mixture of k7 and da - 3 / sec cells gave tumors appearing by 21 days after challenge . however , mice that had been vaccinated twice with 10 6 da - 3 / sec mammary tumor cells had a delay in tumor appearance to 40 days after challenge and a protection against tumor development . indeed , 70 days after challenge less than 50 % of the vaccinated mice had tumors . a possible clue about why the secreted form of muc1 affords protection , was obtained from the analysis of the structures of the muc1 transmembrane and secreted isoforms . a schematic drawing of the cdnas from the muc1 / tm and muc1 / sec isoforms is shown in fig9 . the cdnas are depicted from their 5 ′ termini at the left of the figure . the tandem repeat array is depicted by the barred region and the regions coding for the signal peptide , transmembrane domain , and cytoplasmic domain are indicated by sp , tm , and cyt respectively and have the same shadings in the two forms . the amino acid sequence at the terminal end of the muc1 / sec isoform is vsiglsfpmlp ( seq id no : 1 ) ( 3 ). importantly , a blastp 1 . 4 . 11 ( 6 ) analysis at high stringency of these 11 amino acids revealed approximately 50 % identity with the human ly 49e ligand with no other identity with any known protein sequence . at low stringency there were some similarities with glutathione s - transferase from two echinococcus species , to hypothetical protein mlcb4 . 30 of mycobacterium leprae and to ef hand protein of two plant species ; this peptide is likely to be highly antigenic due to its hydrophilic nature . to ascertain the presence of this peptide in the da - 3 cells transfected with the secreted muc1 isoform , a chicken igy antibody against the unique 11 amino acid peptide bound to klh was prepared . a direct elisa utilizing this reagent was developed to detect the peptide in the supematants of da - 3 / sec cultures . briefly , 50 μl of coating buffer containing 0 . 2 - 10 . 0 μg of protein / ml or tissue culture supernatants from serum free cultures were plated overnight at 4 ° c . blocking buffer was added for 1 hour at 37 ° c ., plates washed 3 times , primary antibody incubated for 1 hour or overnight ; wash 3 times ; add secondary antibody , if required , for 1 hour , wash 3 times ; add visualization solution ; incubate and read . a positive control of purified peptide at known concentrations was employed in all assays . all cultures tested were positive for this molecule ( data not shown ). experiments to evaluate the possible beneficial effects of vaccination with this peptide were performed ( fig1 ). control mice that received injections with da - 3 / tm or da - 3 / neo cells ( 10 6 cells ) resulted in 100 % tumor growth whether in the presence or absence of adjuvant alone . experimental mice received two injections eight days apart with klh - bound unique peptide in the presence of adjuvant . eight days after the second injection the animals were challenged with a mixture of klh bound peptide and tumor cells . challenge with the peptide mixed with da - 3 / tm cells resulted in tumor growth in only 7 out of 12 mice , with an additional animal developing a tumor at 4 weeks . challenge of vaccinated mice with a mixture of klh bound peptide and da - 3 / neo cells afforded even a greater amount of protection and only 40 % of the animals developed tumors , again with one additional mouse showing tumor growth after a month delay . importantly , the survivor animals have shown no signs of tumor at 6 months after original challenge with either da - 3 / tm or da - 3 / neo cells . in further studies , the effect of the peptide vaccination was evaluated in two other tumors . in fig1 a it can be seen that this protocol gave only a small protection and a retardation of tumor appearance when renca cells were implanted in balb / c mice . importantly , when the klh bound immunoenhancing peptide was used in vaccination protocols in another mouse strain ( c57 / bl6 mice ) and challenged with the very aggressive lewis lung carcinoma , protective effects were also observed , as seen in fig1 b . in order to determine whether a lower number of challenge cells mixed with da - 3 sec cells will obviate the necessity of previous exposure to the latter in order to provide protection against tumor growth , 5 × 10 5 da - 3 / tm cells were mixed with an equal amount of da - 3 / sec cells and administered to mice . as shown in fig1 , this treatment provided 60 % protection against tumor development without any previous vaccination . in order to determine which effector cells are involved in the protection observed against da - 3 / sec tumor cell growth , cytotoxicity assays against 51 cr labeled cells were performed using splenic cells from mice that received an inoculation of da - 3 / sec tumor cells two weeks prior to obtaining the cells . 10 × 10 6 splenocytes were cultured for 5 days in the presence of 2 × 10 5 - 2 × 10 6 mitomycin - c ( mmc ) treated da - 3 / sec cells . splenocytes primed with da - 3 / sec exhibited a strong cytolytic activity against da - 3 / sec cells ( fig1 ). da - 3 / sec primed splenocytes have low levels of cytotoxicity against da - 3 / tm , da - 3 / neo and da - 3 targets . these results suggest that a population of lymphocytes is expanding in vitro upon stimulation with da - 3 / sec , which have potent effector function against da - 3 / sec targets . cells of the immune system have multiple mechanisms of killing , which include fas - fas ligand interactions ( 7 ) and perforin - granzyme mediated killing ( 8 ) the da - 3 / sec primed splenocytes were treated with anti - fas antibody or concanamycin a ( cma ), an inhibitor of vacuole acidification through blockage of h + atpase that blocks perforin - mediated killing ( 9 ). cma effectively blocked killing of da - 3 / sec targets in a dose - dependent manner ( fig1 ), while no effect on cytotoxicity was observed with the addition of the anti - fas antibody ( fig1 ). these results indicate that fas / fasl does not play a role in the cytotoxicity of da - 3 / sec cells , but that it is mediated through the perforin - granzyme pathway . in order to determine whether in vivo exposure to da - 3 / sec cells results in higher nk activity against yac - 1 target cells ( a classic murine nk - target cell ) ( 10 ), an experiment was performed in which normal splenocytes were compared with splenocytes that had 3 or 10 day exposure to da - 3 / sec cells in vivo with regard to their ability to induce lysis . as seen in fig1 , splenic cells from balb / c mice injected 3 days prior to assay with da - 3 / sec cells have somewhat higher levels of lytic activity against yac - 1 cells as compared to those from normal mice . this enhanced nk reactivity is more pronounced in mice that have been inoculated with the da - 3 / sec cells 10 days prior testing . other preliminary studies in our laboratory suggested that da - 3 cells transfected with the human muc1 secreted form , but not the transmembrane form , stimulate effector cells to respond against da - 3 / sec cells both in vivo and in vitro . as mentioned above , there is a unique 11 amino acid peptide present on the c - terminus of the muc1 / sec protein , but not on the muc1 / tm protein . a synthetic peptide was made corresponding to this peptide ( termed “ immunoenhancing peptide ”, or iep ), which when used in vaccination protocols , has been found to have protective effects against the da3 , da3 / neo , da3 - tm , k7 osteosarcoma , renca , and lewis lung carcinoma cells . we investigated the possible effects of the peptide on the cytotoxicity reaction . we tested it on yet another tumor model , i . e . the d1 - dmba - 3 mammary tumor bearing mice . the da - 3 cell line was originally derived from this tumor , which is routinely passaged in vivo in our laboratory in the syngeneic balb / c mice . in fig1 , it can be seen that addition of the muc1 / sec unique peptide in vitro to splenocytes from either normal and d1 - dmba - 3 tumor bearing mice for 5 days results in significant levels of cytotoxicity against the da - 3 / sec tumor targets . importantly , vaccination protocols similar to those described above gave significant protection against the growth of the d1 - dmba - 3 tumor . the ability of this peptide to stimulate immune cells in a nk cell assay was tested . this assay was chosen based on the observation that the da - 3 / sec tumor was suppressed during the initial stages of growth in the nude mice , suggesting that an innate immune component may be involved in the immediate reaction against da - 3 / sec . therefore , to test for immunomodulatory activity of the immunoenhancing peptide ( iep ), balb / c splenocytes were pretreated for 30 - 45 minutes with 10 μg iep and subsequently , chromium - labeled yac - 1 or el - 4 targets were added in a 4 - hour assay as previously described ( 11 ). as seen in fig1 , peptide addition resulted in an elevation of cytotoxic levels from normal splenocytes against the classic murine nk - target cells , yac - 1 . furthermore , el - 4 target cells , which are not susceptible to nk - type of cytotoxicity by normal splenic cells , were readily lysable by these effectors , when they were previously exposed to the immunoenhancing peptide . in other experiments we were able to activate splenic effector cells from normal balb / c or c57 bl / 6 ( b6 ) mice to kill da - 3 / sec target cells by previous exposure to the muc1 / sec unique peptide ( fig1 ). it could be argued that the cytotoxicity observed is due to a direct toxic effect of the unique 11 amino acid peptide on the tumor cells . to test this possibility we added high levels of the iep to several tumor targets in vitro and determined the cell viability after 4 days in culture . as seen in table 3 , such treatment with much higher peptide levels than those to which the cells are exposed in vivo , or in the effector cell mediated cytotoxicity assays caused no inhibition of growth or cytocidal activity against the tumor cells . the data disclosed herein indicate that transfection of a novel secreted isoform of the muc1 gene ( muc1 / sec ) prevents the growth of an aggressive , immunogenic established tumor cell line in balb / c mice . when used in vaccination protocols , it can afford protection against syngeneic tumors in a non - specific manner . a unique eleven amino acid peptide present in the muc1 / sec molecule is involved in the protective effects and when used in vaccination protocols it is capable of acting effectively against various tumor cell lines . the data also suggest that cytotoxicity by effector cells of the innate and / or adaptive immune system is involved in the observed results . since the effects of this peptide are not restricted to a given type of tumor or mouse strain , it should be possible to use it alone and / or in combination with other immunomodulatory molecules against a wide variety of tumors . no toxicity has been detected in the animals injected with the peptide . the broad spectrum of action of this iep may permit the use of this agent not only against tumors , but also against viral or bacterial diseases such as hiv , hepatitis b and c , and other microbial infections ( e . g ., tuberculosis ), where an enhancement of the immune responses may have positive effects in the exposed individuals . in describing preferred embodiments of the present invention , specific terminology is employed for the sake of clarity . however , the invention is not intended to be limited to the specific terminology so selected . it is to be understood that each specific element includes all technical equivalents , which operate in a similar manner to accomplish a similar purpose . the embodiments illustrated and discussed in the present specification are intended only to teach those skilled in the art the best way known to the inventors to make and use the invention , and should not be considered as limiting the scope of the present invention . the exemplified embodiments of the invention may be modified or varied , and elements added or omitted , without departing from the invention , as appreciated by those skilled in the art in light of the above teachings . it is therefore to be understood that , within the scope of the claims and their equivalents , the invention may be practiced otherwise than as specifically described . all patents , published patent applications and other published references cited herein are hereby incorporated by reference . references are listed below for convenience . 1 . ceriani r l , thompson k e , peterson j a , and abrahams s : surface differentiation antigens of human mammary epithelial cells carried on the human milk fat globule . proc . natl . acad . sci . ( wash ) 74 : 582 - 587 , 1977 . 2 . lloyd k o , burchell j , kudryashou v , yin b w t , and taylor - papadimitriou j : comparison of o - linked carbohydrate chains in muc1 mucin from normal breast epithelial cell lines and breast carcinoma cell lines . j . biol . chem . 271 : 33325 - 33334 , 1996 . 3 . smorodinsky n , weiss m , hartmann m - l , baruch a , harness e , yaakobovitz m , keydar i , and wreschner d h : detection of a secreted muc1 / sec protein by muc1 isoform specific monoclonal antibodies . biochem . biophys . res . comm . 228 : 115 - 121 , 1996 . 4 . fu y - x , watson ga , kasahara m , and lopez d m : the role of tumor derived cytokines on the immune system of mice bearing mammary adenocarcinomas . i . induction of regulatory macrophages by in vivo administration of rgm - csf . j . immunol . 146 : 783 - 789 , 1991 . 5 . keydar i , chou c s , hareuveni m , tsarfaty i , sahar e , selzer g , chaitchik s , and hizi a : production and characterization of monoclonal antibodies identifying breast tumor associated antigens . proc . natl . acad . sci . 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