Patent Abstract:
a cancer antigen peptide which is a partial peptide derived from parathyroid hormone - related protein , and which is capable of binding to an hla - a24 or hla - a2 antigen and being recognized by cytotoxic t lymphocytes , or a derivative thereof having the functionally equivalent properties , can be useful for the treatment and prevention of prostate cancer .

Detailed Description:
prostate cancer appears to be a good target for the development of specific immunotherapies [ 25 ]. in recent years , the inventors have attempted to identify epitope peptides derived from prostate - related antigens that would be able to generate prostate cancer - reactive ctls from prostate cancer patients [ 22 , 23 , 26 ]. however , one major obstacle encountered when treating prostate cancer patients is the treatment of prostate cancer metastasized to bone tissues . therefore , the inventors undertook the present study to identify antigenic epitopes which could potentially be suitable for specific immunotherapeutic treatment of hla - a24 + or hla - a2 + prostate cancer patients with metastases . in this study , pthrp - derived peptides immunogenic in hla - a24 + or hla - a2 + prostate cancer patients were identified . pthrp 36 - 44 and pthrp 102 - 111 peptides , among four different pthrp peptides carrying the hla - a24 binding motif , and pthrp 42 - 51 and pthrp 59 - 67 peptides , among seven different pthrp peptides carrying the hla - a2 binding motif , efficiently induced peptide - specific cytotoxic t lymphocytes from peripheral blood mononuclear cells ( pbmcs ) of hla - a24 + and hla - a2 + prostate cancer patients , respectively . peptide - stimulated pbmcs showed cytotoxicity against prostate cancer cells in an hla - a24 or hla - a2 - restricted manner . experiments using antibodies and cold inhibition targets confirmed that their cytotoxicity was dependent on pthrp peptide - specific and cd8 + t cells . further , immunoglobulin gs ( iggs ) reactive to the pthrp 102 - 111 or the pthrp 110 - 119 peptide which binds to an hla - a24 molecule and pthrp 42 - 51 peptide which binds to an hla - a2 molecule were frequently detected in the plasma of prostate cancer patients , suggesting that the pthrp 102 - 111 and pthrp 42 - 51 peptides are efficiently recognized by both cellular and humoral immune responses in cancer patients . these results indicate that the peptide of the invention could be a promising target molecule for specific immunotherapy of hla - a24 + or hla - a2 + prostate cancer patients with metastases . in the present invention , new pthrp peptides which have the potential to generate prostate cancer - specific ctls in hla - a24 + or hla - a2 + prostate cancer patients have been identified , and thereby the possibility of creating a peptide vaccine targeting pthrp has been expanded . the inventors revealed that both the pthrp 36 - 44 and pthrp 102 - 111 peptides have the potential to more efficiently induce prostate cancer - reactive ctls in hla - a24 + prostate cancer patients , as well as the pthrp 42 - 51 and pthrp 59 - 67 peptides in hla - a2 + prostate cancer patients . pbmcs from hla - a24 + prostate cancer patients showed peptide - specific ifn - γ production in 7 or 6 of 10 patients when stimulated with the pthrp 36 - 44 peptide or the pthrp 102 - 110 peptide , respectively . hla - a2 - binding pthrp 42 - 51 and the pthrp 59 - 67 peptides induced peptide - specific ifn - γ production in 5 or 4 of 10 patients , respectively . more importantly , pbmcs that were stimulated with these pthrp peptides showed cytotoxicity against prostate cancer cells in an hla - a24 - or hla - a2 - restricted manner . these results indicate that the above pthrp peptides are immunogenic and therefore potentially useful for specific immunotherapy of hla - a24 + or hla - a2 + prostate cancer patients with metastases . those peptides also induced peptide - specific and cancer - reactive ctls from the pbmcs of hla - a24 + or hla - a2 + healthy donors . this result is consistent with that of a previous report demonstrating induction of pthrp peptide - specific ctls from pbmcs of hla - a2 + healthy donors [ 17 ]. as the homology between each of these pthrp peptides and pth is low , cross - reactivity between the pthrp peptides and pth could be excluded . low levels of pthrp have been sporadically detected in keratinocytes , uterus , and mammary glands during lactation [ 27 ]. recent advances in tumor immunology have revealed that self - antigens on human cancer cells are the most prevalent antigens recognized by the immune system [ 2 - 4 ]. ctl precursors reactive to non - mutated self antigens might circulate in the peripheral blood of both certain healthy donors and cancer patients . here , the inventors investigated whether or not iggs against pthrp peptides would be detectable in the plasma from hla - a24 + or hla - a2 + prostate cancer patients , because antibodies against class i - binding cancer antigen peptides had already been observed in certain cancer patients and healthy donors [ 20 , 21 ]. we also previously reported that iggs reactive to peptides derived from prostate - related antigens were frequently detectable in healthy donors and prostate cancer patients [ 22 - 24 ]. in the present invention , iggs reactive to either the pthrp 102 - 111 or pthrp 110 - 119 peptide among the hla - a24 - binding peptides and the pthrp 42 - 51 peptide among the hla - a2 - binding peptides were frequently detected in healthy donors as well as in prostate cancer patients . this means that the pthrp 102 - 111 and pthrp 42 - 51 peptides were efficiently recognized by both the cellular and the humoral immune system . although the roles played by peptide - specific igg in anti - tumor immune responses have not been clear yet , the clinical trials conducted by the inventors revealed that peptide vaccination frequently resulted in the induction of iggs reactive to administered peptides [ 28 , 29 ]. in addition , the induction of iggs reactive to vaccinated peptides was positively correlated with longer survival of advanced lung cancer patients [ 30 ]. as regards the use of peptide vaccination in cases of gastric cancer , prolonged survival has been observed in patients showing not only cellular but also humoral immune responses to vaccinated peptides [ 31 ]. furthermore , the induction of iggs reactive to the administered peptides was correlated with a clinical response among patients with recurrent gynecologic cancer [ 32 ]. interestingly , the levels of igg specific to the pthrp 102 - 111 peptide was lower in patients with advanced hrpc than in those with non - advanced prostate cancer ( unpublished data ). vaccination with the pthrp 102 - 111 peptide into such patients could elicit the induction of peptide - specific igg and might lead to clinical responses . further study could shed light on the role of peptide - specific igg in the anti - tumor immune response . the present invention is further illustrated by the following examples , but is not limited by these examples in any respect . the statistical significance of the data was determined using a two - tailed student &# 39 ; s t - test . a p value of less than 0 . 05 was considered to be statistically significant . identification of candidates to generate peptide - specific ctls from hla - a24 + prostate cancer patients all hla - a24 + prostate cancer patients and hla - a24 + healthy donors were enrolled in this study after informed consent was obtained . the prostate cancer patients # 2 , # 3 , # 5 , and # 9 were associated with bone metastases . none of these participants was infected with hiv . twenty milliliters of peripheral blood was obtained , and pbmcs were prepared by ficoll - conray density gradient centrifugation . the expression of hla - a24 molecules on the pbmcs of cancer patients and healthy donors was determined by flow cytometry . c1r - a24 is an hla - a * 2402 - expressing subline of c1r lymphoma ( oiso m , eura m , katsura f , takiguchi m , sobao y , masuyama k , nakashima m , itoh k , ishikawa t . a newly identified mage - 3 - derived epitope recognized by hla - a24 - restricted cytotoxic t lymphocytes . int . j . cancer 81 : 387 - 394 , 1999 ). all cell lines were maintained in rpmi - 1640 medium ( gibco brl , grand island , n . y .) supplemented with 10 % fcs . four pthrp - derived peptides ( listed in table 1 hereinafter ) were prepared based on their binding affinity to hla - a24 molecules , hla - a24 binding motif [ 18 , 19 ]. all peptides were of & gt ; 90 % purity and were purchased from biologica co ., nagoya , japan . influenza ( flu ) virus - derived ( rfyiqmcyel ), ebv - derived ( tygpvfmcl ), and hiv - derived peptides ( rylrqqllgi ) with the hla - a24 binding motif were used as controls . all peptides were dissolved with dmso at a dose of 10 mg / ml . although the pthrp 1 - 36 peptide is a propeptide [ 35 ], the pthrp 25 - 34 peptide was included in the sequence . as regards the difference in amino acids , three amino acids were found to differ between the pthrp 36 - 44 peptide and pth , and all of the amino acids were found to differ between the other three pthrp peptides and pth . to investigate the immunogenicity of these four pthrp peptides , the pbmcs of 10 hla - a24 + healthy donors and 10 hla - a24 + prostate cancer patients were stimulated with each of the four pthrp peptides and were examined for their ifn - γ production in response to c1r - a24 cells , which were pre - pulsed with either a corresponding pthrp peptide or the hiv peptide . flu - and ebv - derived peptides were used as controls . the assay for detection of peptide - specific ctls in pbmcs was performed according to a previously reported method [ 34 ]. in brief , pbmcs ( 1 x 10 5 cells / well ) were incubated with 10 pg / mi of each peptide in a u - bottom - type 96 - well microculture plate ( nunc , roskilde , denmark ) at a volume of 200 μl of culture medium . the culture medium consisted of 45 % rpmi - 1640 , 45 % aim - v medium ( gibco brl ), 10 % fcs , 100 u / ml of il - 2 , and 0 . 1 mm mem nonessential amino acid solution ( gibco , brl ). half of the culture medium was removed and replaced with new medium containing a corresponding peptide ( 20 μg / ml ) every 3 days . on the 15th day of culture , the cultured cells were separated into 4 wells , and two wells were used for pthrp peptide - pulsed c1r - a24 cells , and the other two wells were used for the hiv peptide - pulsed c1r - a24 cells . after an 18 - hr incubation period , the supernatants were collected , and the level of ifn - γ was determined by elisa ( limit of sensitivity : 10 pg / ml ). the assay was carried out in quadruplicate , and the mean of two wells which show the highest significance compared to the control is shown in table 1 . successful induction of peptide - specific ctls was judged to be positive when significant values ( p & lt ; 0 . 05 by two tailed student &# 39 ; s t - test ) were observed . as a result , the pthrp 36 - 44 , pthrp 102 - 111 , pthrp 25 - 34 , and pithrp 110 - 119 peptides induced peptide - specific ctls in 6 , 5 , 2 , and 3 of 10 hla - a24 + healthy donors , respectively . these pthrp peptides also induced peptide - specific ctls in 7 , 6 , 3 , and 1 of 10 hla - a24 + prostate cancer patients , respectively . these findings indicate that the pthrp 36 - 44 and the pthrp 102 - 111 peptides are , among others , promising candidate peptides to generate peptide - specific ctls from hla - a24 + prostate cancer patients . induction of prostate cancer - reactive ctls that are hla - a24 - restricted and specific for the pthrp 36 - 44 or pthrp 102 - 111 peptide in order to investigate the hla - a24 - restricted and prostate cancer - reactive cytotoxicity of peptide - stimulated pbmcs , an hla - a24 - expressing lncap cell line , which was designated as lncap - a24 , was prepared . the lncap is an hla - a24 negative prostate cancer cell line . to establish lncap cells stably expressing hla - a24 molecules ( designated as lncap - a24 ), a pcdna3 . 1 / hygro vector ( invitrogen , calif . ), which was inserted with the hla - a * 2402 gene , was electroporated into the lncap cell line ( atcc number : crl - 1740 ), and selection was carried out with hygromycin b ( invitrogen ) at a dose of 170 μg / ml . all cell lines were maintained in rpmi - 1640 medium ( gibco brl , grand island , n . y ., usa ) supplemented with 10 % fcs . lncap has previously been reported to produce pthrp ( 17 ). a parental lncap cell line was negative for the cell surface expression of hla - a24 molecules , whereas the lncap - a24 cell line expressed hla - a24 molecules on their cell surface . flow cytometric analysis was performed on lncap and lncap - a24 cells . these cells were stained with anti - hla - a24 mab , followed by fitc - conjugated anti - mouse igg mab . the dotted lines represent staining without the first rnab . the results of flow cytometric analysis for lncap and lncap - a24 are shown in fig1 . it was then determined whether pbmcs stimulated by either the pthrp 36 - 44 or the pthrp 102 - 111 peptide could induce prostate cancer - reactive ctls from hla - a24 + healthy donors and prostate cancer patients . pbmcs from hla - a24 + healthy donors and prostate cancer patients were repeatedly stimulated with these pthrp peptides . on day 15 , half of the cultured cells were harvested , pooled from 4 wells , and cultured with c1r - a24 cells , which were pre - pulsed with the hiv peptide ( open symbol ) and the indicated pthrp peptide ( closed symbol ) for 18 - hr . the levels of ifn - γ in the supernatants were then determined by elisa . as a result , the pthrp peptide - stimulated pbmcs from hd # 2 , pt # 1 , and pt # 2 produced higher levels of ifn - γ in response to the corresponding pthrp peptide - pulsed c1r - a24 cells than to hiv peptide - pulsed c1r - a24 cells ( fig2 a ). after confirmation that these peptide - stimulated cells could produce ifn - γ in response to pthrp peptide - pulsed c1r - a24 cells , these peptide - stimulated pbmcs were examined for their cytotoxicity against three targets , lncap cells ( hla - a24 − ), lncap - a24 cells ( hla - a24 + ), and pha - blastoid t cells ( hla - a24 + ). specifically , after in vitro stimulation with the pthrp peptides , the peptide - stimulated pbmcs were additionally cultured with 100 u / ml il - 2 for approximately 10 days , in order to obtain a sufficient number of cells to carry out a cytotoxicity assay . then , the obtained cells were tested for cytotoxicity against both lncap and lncap - a24 , and pha - blastoid t cells by a 6 - hr 51 cr - release assay . two thousands of 51 cr - labeled cells per well were cultured with effector cells in 96 - well round - bottomed plates at the indicated effector / target ratios . the results are also shown in fig2 a . the pthrp peptide - stimulated pbmcs from hd # 2 , pt # 1 , and pt # 2 also showed higher levels of cytotoxicity against the lncap - a24 cell line than against the lncap line and hla - a24 + pha - induced t cell blasts . then , in some experiments , either of anti - hla class i ( w6 / 32 : mouse igg2a ), anti - hla - dr ( l243 : mouse igg2a ), anti - cd4 ( nu - th / i : mouse igg1 ), anti - cd8 ( nu - ts / c : mouse igg2a ), or anti - cd 14 ( h 14 : mouse igg2a ) mab was added to the wells at a dose of 20 μg / ml of at the initiation of the assay . as a result , their cytotoxicity against the lncap - a24 was significantly inhibited by the addition of anti - hla - class i or anti - cd8 mabs , but not by the addition of other anti - hla - class ii , anti - cd4 , or anti - cd 14 mab ( fig2 b ). furthermore , the specificity of pthrp peptide - stimulated pbmcs was confirmed by a cold inhibition assay . in brief , 51 cr - labbeled target cells ( 2 × 10 3 cells / well ) were cultured with the peptide - stimulated pbmcs ( 4 × 10 4 cells / well ) in 96 - well round - bottomed plates with 2 × 10 4 cold target cells . c1r - a24 cells , which were pre - pulsed with either the hiv peptide or a corresponding pthrp peptide , were used as cold targets . their cytotoxicity against the lncap - a24 cell line was significantly suppressed by the addition of the corresponding pthrp peptide - pulsed c1r - a24 cells , as a cold target , but this suppression was not observed with the addition of hiv peptide - pulsed c1r - a24 cells ( fig2 c ). these results indicate that both the pthrp 36 - 44 and pthrp 102 - 111 peptides have the potential to induce prostate cancer - reactive ctls from hla - a24 + prostate cancer patients , and that their cytotoxicity against prostate cancer was dependent on pthrp peptide - specific cd8 + t cells . the inventors previously reported that iggs reactive to ctl epitope peptides were detected in healthy donors and patients with various types of cancers [ 20 , 21 ]. iggs reactive to prostate - related antigens were also detected in healthy donors and prostate cancer patients [ 22 - 24 ]. it was examined , therefore , whether igg reactive to the four pthrp - derived peptides , pthrp 36 - 44 , pthrp 102 - 111 , pthrp 25 - 34 , and pthrp 110 - 119 , could be detected in the plasma of cancer patients and healthy donors . peptide - specific igg levels in the plasma were measured by elisa , as previously reported [ 20 , 21 ]. in brief , peptide ( 20 μg / well )- immobilized plates were blocked with block ace ( yukijirushi , tokyo , japan ) and washed with 0 . 05 % tween20 - pbs , after which 100 μl / well of plasma sample diluted with 0 . 05 % tween20 - block ace was added to the plate . after 2 hours incubation at 37 ° c ., the plates were washed and further incubated for 2 hours with a 1 : 1000 - diluted rabbit anti - human igg ( γ - chain - specific ) ( dako , glostrup , denmark ). the plates were washed , and then 100 μl of 1 : 100 - diluted goat anti - rabbit igg - conjugated horseradish peroxidase ( envision , dako ) was added to each well , and the plates were then incubated at room temperature for 40 min . after the plates were washed once , 100 μl / well of tetramethyl benzidine substrate solution ( kpl , guildford , uk ) was added , and the reaction was stopped by the addition of 1 m phosphoric acid . the values are shown as optical density ( od ) units / ml , and responses to the hiv peptide were subtracted . igg reactive to a corresponding pthrp peptide was judged to be positive when the difference of the od in 1 : 100 - diluted plasma exceeded 0 . 05 . the results that igg reactive to either the pthrp102 - 111 or the pthrp 109 - 119 peptide was detected in 8 of 10 healthy donors and in 7 of 10 prostate cancers are shown in table 2 hereinafter . the results are also shown in fig3 a . to confirm the specificity of igg to the indicated pthrp peptide , 100 μl of sample plasma from either hd # 1 and pt # 6 was cultured in a plate pre - coated with either a corresponding pthrp peptide or an irrelevant pthrp peptide . thereafter , the levels of igg reactive to the pthrp 102 - 111 peptide or the pthrp 110 - 119 peptide in the resulting supernatants were determined by elisa . as a result , the levels of pthrp peptide - specific igg were significantly diminished by culturing the plasma in the corresponding pthrp peptide - coated wells ( fig3 b ). this peptide - specific absorption demonstrated the validity of the present assay system . on the other hand , igg reactive to the pthrp 36 - 44 peptide was detected in 3 of 10 healthy donors and 1 of 10 prostate cancer patients , respectively . no igg reactive to the pthrp 25 - 34 was detected in any of the healthy donors or cancer patients ( table 2 and fig3 a ). in a similar way to example 1 , pthrp - derived peptides capable of inducing peptide - specific ctls in hla - a2 + prostate cancer patients were identified . the cancer patients # 1 , # 3 , and # 10 were associated with bone metastases . as the target cells , t2 cell ( atcc deposition number : crl - 1992 ) which is a t and b lymphoblasts - fused cell expressing hla - a * 0201 molecule ( immunogenetics , 21 , 235 - 246 , 1985 ) was used . seven pthrp - derived peptides having a hla - a2 binding motif were prepared ( listed in table 3 ). although pthrp59 - 68 and pthrp165 - 173 peptides had been reported to generate specific ctls from hla - a * 0201 healthy donors [ 17 ], their predicted binding scores were less than 1 in the present study ( table 3 ). the numbers of the amino acid residues shared between pth and the pthrp peptides were as follows : pthrp 42 - 51 ( 5 ), pthrp 43 - 51 ( 4 ) , pthrp 51 - 60 ( 2 ) , pthrp 59 - 67 ( 1 ) , pthrp 103 - 111 ( 0 ). table 3 shows that the results of peptide - specific ctl induction by those peptides in 10 hla - a2 + healthy donors and 10 hla - a2 + prostate cancer patients . pthrp42 - 51 and pthrp59 - 67 peptides induced peptide - specific ctls in 7 and 4 of 10 hla - a2 + healthy donors , respectively . although the reported pthrp59 - 68 peptide is different from the pthrp59 - 67 peptide in only one amino acid , no peptide - specific ctls were induced when pbmcs from the hla - a2 + healthy donors were stimulated with the pthrp59 - 68 peptide . the reported pthrp165 - 173 induced peptide - specific ctls in 4 of 10 hla - a2 + healthy donors . pthrp42 - 51 and pthrp59 - 67 peptides induced peptide - specific ctls in 5 and 4 of 10 hla - a2 + prostate cancer patients , respectively . the reported pthrp59 - 68 and pthrp165 - 173 peptides induced peptide - specific ctls in 5 and 3 of 10 hla - a2 + prostate cancer patients , respectively . the most cases of successful induction of peptide - specific ctls were different when stimulated with either the pthrp59 - 67 or pthrp59 - 68 peptide . in total , these findings indicate that both the pthrp42 - 51 and pthrp59 - 67 peptides are particularly useful new candidate peptides for generating peptide - specific ctls from hla - a2 + prostate cancer patients . induction of prostate cancer - reactive ctls that are hla - a2 - restricted and specific for pthrp 42 - 51 or pthrp 59 - 67 peptide in a similar way to example 2 , it was examined whether or not pthrp 42 - 51 and pthrp 59 - 67 peptides could induce cancer - reactive ctls in pbmcs derived from hla - a2 + healthy donors and prostate cancer patients . an hla - a2 - expressing pc93 prostate cancer cell line was prepared by constitutively expressing an hla - a * 0201 molecule on a pc93 cell which was a hla - a2 - prostate cancer cell line ( established by dr . k . ohnishi , department of urology , kyoto university ) ( pc93 - a2 cell ). the expression of the hla - a * 0201 molecule on the cell line was previously reported [ 24 ]. in addition , the pc93 cells produced pthrp at a level of 2 . 2 pmol / l ( 1 × 10 6 cell / ml for 24 hr ). it was confirmed that pbmcs stimulated with pthrp 42 - 51 and pthrp 59 - 67 peptides could produce ifn - γ in response to the corresponding pthrp peptide - pulsed t2 cells ( fig4 a ). cytotoxicities of these peptide - stimulated pbmcs against pc93 cells , pc93 - a2 cells , and pha - stimulated t cell blasts are shown in fig4 b . fig5 shows the results of examining the cytotoxicity of the peptide - stimulated pbmcs with ( a ) blocking antibodies and ( b ) the cold inhibition assay . these results indicate that the pthrp42 - 51 and pthrp59 - 67 peptides have the potential to induce prostate cancer - reactive ctls in hla - a2 + prostate cancer patients , and that their cytotoxicity is dependent on peptide - specific cd8 + t cells . in a similar way to example 3 , it was examined whether iggs reactive to peptides of pthrp 42 - 51 and pthrp 59 - 67 could be detected in the plasma of 10 healthy donors and 10 cancer patients . the cut - off value at 1 : 100 - diluted plasma was odo . 054 . the result is shown in table 4 . igg reactive to the pthrp 42 - 51 peptide was detected in 8 of 10 healthy donors , and in 7 of 10 prostate cancer patients . igg reactive to the pthrp59 - 67 peptide was detected in 2 of 10 healthy donors , and in 2 of 10 prostate cancer patients . the representative results of 2 healthy donors ( hd # 1 and hd # 6 ) and 2 cancer patients ( pt # 1 and pt # 2 ) are shown in fig6 a . peptide specificities of the iggs were examined by absorption with the corresponding peptide and the results are shown in fig6 b . those results indicate that those peptides , among others pthrp42 - 51 peptide , induce both of cellular and humoral immune responses in caner patients . the inventers identified new two pthrp - derived peptides that are immunogenic in hla - a24 + or hla - a2 + prostate cancer patients . the frequencies of the hla - a24 allele are relatively high throughout the world [ 33 ]. in addition , most caucasians are hla - a * 2 101 positive . on the other hand , hla - a2 subtypes vary considerably in japanese . pthrp - derived peptides were prepared based on the binding motif to an hla - a * 0201 molecule , and both the t2 cells and pc93 - a2 cells express hla - a * 0201 molecules . however , the peptides of the invention also induced peptide - specific ctls from patients with other hla - a2 subtypes , including hla - a * 0206 ,- a * 0207 , and - a * 02 10 , as shown in table3 . the present invention increases the possibility of treating hla - a24 + and hla - a2 + prostate cancer patients with metastases using peptide - based immunotherapy . 1 . greenlee , r . t ., murray , t ., bolden , s ., and wingo , p . a ., cancer statistics 2000 . ca cancer j . clin . 2000 . 50 : 7 - 33 . 2 . boon , t ., coulie , p . g ., and van den eynde , b ., cancer antigens recognized by t cells . immunol . today 1997 . 81 : 267 - 268 . 3 . rosenberg , s . a ., a new era for cancer immunotheraphy based on the genes that encode cancer antigens . immunity 1999 . 10 : 281 - 287 . 4 . renkvist , n ., castelli , c ., robbins , p . f ., and parmiani , g ., a listing of human cancer antigens recognized by t cells . cancer immunol . immunother . 2001 . 50 : 3 - 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