Patent Abstract:
telomerase peptides that bind mhc are disclosed . the instant application also discloses vaccines containing said peptides and methods of using said peptides to enhance a ctl response against mammalian cancer cells .

Detailed Description:
as used herein , the terms “ telomerase ” and “ telomerase complex ” refer to functional telomerase enzymes . it is intended that the terms encompass the complex of proteins found in telomerases . for example , the terms encompass the 123 kda and 43 kda telomerase protein subunits . telomerase is a ribonucleoprotein enzyme , which has been linked to malignant transformation in human cells . telomerase activity is increased in the vast majority of human tumors making its gene product the first molecule common to all human tumors . the generation of endogenously - processed telomerase peptides bound to class i major histocompatibility complex ( mhc ) molecules could therefore target cytotoxic t lymphocytes ( ctl ) to tumors of different origins . this could advance vaccine therapy against cancer provided that precursor ctl recognizing telomerase peptides in normal adults and cancer patients can be expanded through immunization . applicant demonstrates here that the majority of normal individuals and patients with prostate cancer immunized in vitro against two hla - a2 . 1 restricted peptides from telomerase reverse transcriptase ( htrt ), develop htrt specific ctl . this suggests the existence of precursor ctl for htrt in the repertoire of normal individuals and in cancer patients . most importantly , cancer patients &# 39 ; ctl specifically lysed a variety of hla - a2 + cancer cell lines , demonstrating immunological recognition of endogenously - processed htrt peptides . moreover , in vivo immunization of hla - a2 . 1 transgenic mice generated a specific ctl response against both htrt peptides . based on the induction of ctl responses in vitro and in vivo , and the susceptibility to lysis of tumor cells of various origins by htrt ctl , applicant suggests that htrt could serve as a universal cancer vaccine for humans . telomerase is a unique ribonucleoprotein that mediates rna - dependent synthesis of telomeric dna ( 1 ), the distal ends of eukaryotic chromosomes that stabilize the chromosomes during replication ( 2 , 3 ). when activated , telomerase synthesizes telomeric dna and compensates for its loss with each cell division ( 4 ). since telomeres shorten progressively with successive cell divisions , telomere length is considered to mirror the replicative history of cell lineage ( 5 ) and cell population dynamics ( 6 , 7 ). in mice , telomerase appears to play an essential role in the long - term viability of high - renewal organ systems such as the reproductive and haemopoietic systems ( 8 ). maintenance of a constant telomere length ensures chromosomal stability , prevents cells from aging , and confers immortality ( 9 - 11 ). mice lacking telomerase rna show that telomerase activation is a key event in malignant cell transformation ( 8 , 12 , 13 ). in humans , in vitro studies show that the long - term ectopic expression of telomerase reverse transcriptase ( htrt ) in normal fibroblasts is sufficient for immortalization but not malignant transformation ( 14 ). however , the expression of htrt in combination with two oncogenes ( sv40 t antigen and ras ) promotes tumor transformation in normal human epithelial and fibroblast cell lines ( 15 ). these transformed cells form tumors in nude mice . thus , although telomerase per se is not tumorigenic , it plays a direct role in oncogenesis by allowing pre - cancerous cells to proliferate continuously and become immortal . the pcr - based trap assay ( 16 ) reveals a striking correlation (& gt ; 80 %) between high telomerase activity and tumors of different histological origins and types ( 17 , 18 ). in contrast , normal tissues display little or no telomerase activity ( 18 , 19 ). therefore , telomerase expression in tumors is much greater than her2 / neu and mutated p53 , which range between 30 % and 50 % respectively ( 20 , 21 ). from the foregoing , it is reasonable that expression of htrt in cancer cells is a likely source of peptides that , upon association with major histocompatibility complex ( mhc ) class i molecules , could target cytotoxic t lymphocytes ( ctl ) to cancer cells . an interesting analogy exists with hiv - 1 reverse transcriptase , an enzyme similar to htrt , which gives origin to peptide / mhc class i complexes that target ctl responses to virus infected cells ( 22 ). thus , since high telomerase activity is widespread among human tumors , htrt could serve as a universal tumor antigen for immunotherapy and vaccine approaches . htrt is encoded in the genome and is in all respects a self antigen . consequently , cd8 + t lymphocytes with a receptor for mhc / htrt peptide complexes are expected to be eliminated during thymic negative selection , reducing the potential precursor t cell repertoire and imposing limitations on their expansion upon encounter with tumor cells in adult life . additionally , stimulation by antigen in the absence of a second signal induces clonal anergy ( 23 ), further hampering the potential repertoire . the extent to which these events affect the normal adult repertoire , and whether or not exposure to htrt during cancer formation has any adverse effect on the ability of cancer patients to respond , is not known . because answering these questions is relevant to future strategies of immune intervention targeted at htrt , the ability of normal individuals and cancer patients to mount a ctl response in vitro against two htrt peptides restricted by the hla - a2 allele was analyzed . htrt synthetic peptides p540 ( 540ilakflhwl548 , seq id no : 1 ), p865 ( 865rlvddfllv873 , seq id no : 2 ) and mart - 1 ( 27aagigiltv35 , seq id no : 3 ) were purchased from the biopolymer synthesis center ( caltech , pasadena , calif .). synthetic peptides 128tppayrppnapil140 ( seq id no : 4 ) of the hepatitis b core antigen ( hbvc ), 571ylsganlnl579 ( seq id no : 5 ) of carcinoembryonic antigen ( cea ), 476vlyrygsfsv486 ( seq id no : 6 ) of melanoma antigen gp100 , 476ilkepvhgv484 ( seq id no : 7 ) of hiv - 1 reverse transcriptase were purchased from neosystem ( strasburg , france ). buffy coats from normal donors were purchased from the san diego blood bank . hla - a2 + individuals were selected by facs screening using monoclonal antibody bb7 . 2 . prostate cancer patients were recruited through the division of urology ( university of california , san diego ). blood from these patients was obtained by venipuncture . hla - a2 + individuals were selected by facs screening using monoclonal antibody bb7 . 2 . blood collection and experiments were performed in accordance with an approved irb . t2 cells were a kind gift of dr . peter creswell ( yale university ). melanoma cell lines 624 and 1351 were the kind gift of dr . john wunderlich ( national cancer institute , bethesda , md .). prostate cancer cell lines lncap and pc - 3 were the kind gift from dr . antonella vitiello ( pri johnson , la jolla calif .). breast , colon and lung tumor cell lines were obtained from atcc , rockville , md . pbmc were separated by centrifugation on ficoll - hypaque gradients and plated in 24 - well plates at 5 × 10 5 cells / ml / well in rpmi - 1640 supplemented with 10 % human ab + serum , l - glutamine and antibiotics ( cm ). autologous pbmc ( stimulators ) were pulsed with htrt synthetic peptides p540 or p865 ( 10 μg / ml ) for 3 hours at 37 ° c . cells were then irradiated at 5000 rads , washed once , and added to the responder cells at a responder : stimulator ratio ranging between 1 : 1 and 1 : 4 . the next day , 12 iu / ml il - 2 ( chiron co ., emeryville , calif .) and 30 iu / ml il - 7 ( r & amp ; d systems , minneapolis , minn .) were added to the cultures . lymphocytes were re - stimulated weekly with peptide - pulsed autologous adherent cells as follows . first , autologous pbmc were incubated with htrt peptide ( 10 μg / ml ) for 3 hours at 37 ° c . non - adherent cells were then removed by a gentle wash and the adherent cells were incubated with fresh medium containing the htrt peptide ( 10 μg / m ) for an additional 3 hours at 37 ° c . second , responder cells from a previous stimulation cycle were harvested , washed and added to the peptide - pulsed adherent cells at a concentration of 5 × 10 5 cells / ml ( 2 ml / well ) in medium without peptide . recombinant il - 2 and il - 7 were added to the cultures on the next day . hhd mice were immunized subcutaneously at the base of the tail with 100 μg of individual htrt peptide emulsified in incomplete freunds &# 39 ; adjuvant ( ifa ). half of the mice were immunized with the htrt peptide and 140 μg of the helper peptide tppayrppnapil ( seq id no : 4 ), which corresponds to residues 128 - 140 of the hepatitis b core antigen ( hbvc ) ( 25 ). the relative avidity was measured as previously described ( 25 ). briefly , t2 cells were incubated overnight at 37 ° c . in rpmi supplemented with human β2 - microglobulin ( 100 ng / ml ) ( sigma , st . louis , mo .) in the absence ( negative control ) or presence of the test peptide or the reference peptide 476ilkepvhgv484 ( seq id no : 7 ) of hiv - 1 reverse transcriptase at various final peptide concentrations ( 0 . 1 - 100 μm ). cells were incubated with brefeldin a ( 0 . 5 μg / ml ) for one hour and subsequently stained with a saturating concentration of monoclonal antibody bb7 . 2 for 30 minutes at + 4 ° c . followed by washing and a second incubation with a goat antibody to mouse ig ( fab ′) 2 conjugated to fitc ( caltag , south san francisco ). cells were then washed , fixed with 1 % paraformaldehyde and analyzed in a facs calibur cytofluorimeter ( becton & amp ; dickinson , san jose , calif .). the mean fluorescence intensity of each concentration minus that of cells without peptide was used as an estimate of peptide binding . results are expressed as values of ra , which is the ratio of the concentration of test peptide necessary to reach 20 % of the maximal binding by the reference peptide over that of the reference peptide so that the lower the value the stronger the binding . dissociation of the test peptide from the hla - a2 . 1 molecule reflects the half - life of fluorescence intensity of the peptide / mhc complex over time . the half - life of the complex ( dc50 ) refers to the time ( hours ) required for a 50 % reduction of the to mean fluorescence intensity ( 25 ). synthetic peptides 571ylsganlnl579 ( seq id no : 5 ) of carcinoembryonic antigen ( cea ) and 476vlyrygsfsv486 ( seq id no : 6 ) of melanoma antigen gp100 were used as internal controls to account for inter - tests variability and for consistency with previously reported ra and dc50 measures ( 25 ). ( a ) the induction of ctl in human pbmc was monitored in a conventional 51 cr - release assay . briefly , peptide - pulsed tap -/ hla - a2 . 1 + human t2 cells were incubated with 10 μg of htrt peptides or with the mart - 1 control peptide for 90 minutes during labeling with 51 cr . after washing , the target cells were added to serially diluted effectors in 96 - well microplates . after a 6 hour incubation period at 37 ° c ., supernatants were harvested and counted in a trilux betaplate counter ( wallac , turku , finland ). results are expressed as the percentage (%) of specific lysis and determined as follows : [( experimental cpm − spontaneous cpm )/( maximum cpm spontaneous cpm )]× 100 . ( b ) the induction of ctl in hhd mice was assessed as follows . spleen cells were harvested 7 days after immunization and were restimulated in vitro with the corresponding htrt peptide and lps ( 25 μg / ml )- stimulated irradiated ( 5000 rads ) syngeneic spleen cells . after six days of culture the cells were harvested and tested for their ability to lyse hhd - transfected / tap - rma - cells in a 4 hour 51 cr - release assay ( 25 ). specific lysis was calculated as indicated in the legend of fig1 . values refer to maximal cytotoxicity measured for individual responder mice at an effector to target ratio of 60 : 1 . the amino acid sequence of htrt ( locus af015950 ) ( 19 ) was analyzed for 9mer peptide sequences containing known binding motifs for the hla - a2 . 1 molecule [ 52 ; 35 ; 60 ], a subtype encompassing 95 % of hla - a2 allele , which is expressed in about 50 % of the caucasian population ( 26 - 28 ). peptides were identified by reverse genetics based on canonical anchor residues for hla - a2 . 1 ( 29 ), and by using the software of the bioinformatics & amp ; molecular analysis section ( nih ) website bimas . dcrt . nih . gov / molbio / hla_bind / index . html , which ranks 9mer peptides on a predicted half - time dissociation coefficient from hla class i molecules ( 30 ). from an initial panel of 30 candidate peptides applicant retained two sequences , 540ilakflhwl548 ( seq id no : 1 ) and 865rlvddfllv873 ( seq id no : 2 ), denoted hereunder as p540 and p865 . since the immunogenicity of mhc class i - restricted peptides reflects to some degree their binding and stabilizing capacity for mhc class i molecules ( 31 - 33 ) applicant sought direct proof of the strength of interaction between the two htrt peptides and the hla - a2 . 1 molecule in a conventional binding / stabilization assay that uses the antigen - transporting deficient ( tap -) hla - a2 . 1 + human t2 cells . the relative avidity ( ra ) calculated in reference to 476ilkepvhgv484 ( seq id no : 7 ) of hiv - 1 reverse transcriptase , a canonical high binder peptide ( 25 ), was 2 . 9 and 2 . 5 for p540 and p865 , respectively ( table i ). the stability of each peptide bound to hla - a2 . 1 was measured as the half - life of the complex a . the relative avidity of htrt peptides was measured relative to the reference peptide ilkepvhgv ( seq id no : 7 ) at a final peptide concentration of 0 . 1 - 100 mm . b . dc50 refers to the time required for a 50 % reduction in mean fluorescence intensity . c . peptides of human carcinoembryonic antigen ( cea ) ( p571 ) and human melanoma antigen gp100 ( p476 ) were used as internal controls for comparison with previously reported values 33 . ( dc50 ) and was in the order of 4 - 6 hours for p540 and 2 - 4 hours for p865 , respectively . collectively , these measurements indicate that both htrt peptides are excellent binders to hla - a2 . 1 albeit p865 has a faster dissociation rate . the presence of precursor t cells for both htrt peptides and their expansion upon antigen stimulation were tested using peripheral blood lymphocytes ( pbmc ) of 10 hla - a2 + normal blood donors in an in vitro immunization assay . nine out of 10 individuals responded to immunization generating t cells that lysed peptide - pulsed t2 cells as targets starting from the third round of peptide stimulation . all nine responders generated ctl specific for p540 and seven responded against p865 ( fig1 , a and b ). the values of maximal lysis varied from individual to individual and ranged between 28 - 68 % and 20 - 68 %, respectively . in two instances ( donor 975 and 980 ) there was a lower but measurable non - specific lysis , possibly due to contaminant nk cells . thus , by random testing of normal hla - a2 + individuals , it was clearly established that both htrt peptides are immunogenic , implying that precursor ctl for htrt are present in the peripheral adult repertoire . whether or not ctl against htrt could also be induced in cancer patients was studied in four hla - a2 . 1 + individuals with clinical and histological diagnosis of prostate cancer . all four patients were refractory to hormonal therapy , three had metastases and none had prostatectomy . in prostate cancer , the most common cause of cancer in men , high htrt expression has been documented in 84 % of cases ( 34 ). marked lysis of peptide - pulsed t2 cells was observed in 3 out of 4 individuals after three rounds of in vitro stimulation ( fig2 , a and b ). both peptides yielded comparable ctl responses in all three individuals with maximal lysis ranging between 27 - 49 % and 48 - 52 %, respectively . ctl against both peptides lysed lncap , a hla - a2 . 1 + prostate cancer cell line , with maximal lysis ranging between 24 - 36 % for p540 and 12 - 40 % for p865 . prostate cancer cell line pc - 3 , which is hla - a2 . 1 −, was used as control and was not lysed ( fig2 , c ). both prostate cancer cell lines tested positive for htrt by the trapeze ( telomerase detection assay ; intergen ) ( not shown ), suggesting that the ctl generated against the synthetic peptides might lyse cancer cells by recognizing htrtpeptide / mhc class i complex at the surface of cancer cells . cold target competition experiments were performed in an attempt to understand if lysis of the lncap tumor cell line was specific for endogenously processed htrt peptides . in these experiments the lysis of lncap cells by ctl from a prostate cancer patient was competed for by t2 cells pulsed in vitro with p540 or p865 ( 10 μg / ml ). peptide - loaded t2 cells caused a dose - dependent inhibition of lysis of lncap cells in both peptide combinations ( fig3 , a ). applicant further assessed the specificity of the ctl generated against each one of the two htrt peptides by testing them on t2 targets pulsed with irrelevant hla - a2 binding peptides . neither t2 cells pulsed with peptide 27aagigiltv35 ( seq id no : 3 ) from the melanoma antigen mart - 1 nor t2 cells pulsed with a non - homologous htrt peptide were lysed ( fig3 , b ). collectively , these studies show that 1 ) patients &# 39 ; ctl are specific for the htrt peptide used to induce them , and 2 ) lysis of prostate cancer cells is mediated by , and is specific for , endogenously - processed htrt peptides complexed with hla - a2 . 1 molecules , suggesting chemical identity between naturally processed peptides on tumor cells and the synthetic peptides used for immunization . formal validation will require elution of peptides from tumor cells and their analysis by tandem mass spectrometry ( 35 ). studies on mhc restriction were performed using blocking antibodies . lysis of peptide - pulsed t2 cells by ctl lines generated from a prostate cancer patient was inhibited by the anti - mhc class i monoclonal antibody bb7 . 2 in both peptide combinations ( fig3 ), but not by the anti - mhc class ii monoclonal antibody q5 / 13 ( 36 ) nor by transfectoma antibody 1rgd3 that blocks nk cells ( 37 ). by two - color facs analysis , the phenotype of t cells proliferating after three rounds of in vitro stimulation with htrt peptide was cd3 + ( 78 %), cd8 + ( 37 %), cd4 + ( 36 %) and cd16 / 56 ( 6 %). collectively , these experiments confirm that effector t cells generated by in vitro immunization are mhc class i - restricted ( cd8 +) t cells which do not possess nk activity . htrt is expressed in normal cells such as circulating b and t cells , germinal center b cells , thymocytes and cd34 + progenitor hemopoietic cells ( 6 , 7 , 38 ). this implies that ctl generated against htrt peptides could engender an autoimmune attack on normal cells . to this end , applicant verified whether cancer patients &# 39 ; ctl would lyse hla - a2 + cd34 + cells . neither ctl against p540 nor those against p865 induced any lysis over a wide range of effector to target ( e : t ) ratios ( not shown ). thus , at least with respect to hemopoietic stem cells an autoimmune attack appears unlikely . this is consistent with the fact that activated t cells were not lysed by htrt ctl in culture . whether peptides can serve as immunogens in vivo and elicit a ctl response depends on a variety of factors such as the mode of immunization , suitable activation of antigen presenting cells , the frequency of precursor cells , and binding and stabilization of mhc class i molecules by peptide . in this study applicant demonstrated ( table i ) that both peptides bind to hla - a2 . 1 with a ra & lt ; 3 but possess different dissociation rates . in either case applicant was able to generate ctl responses in vitro from pbmc of normal blood donors as well as prostate cancer patients . therefore , a reasonable expectation would be that they may also be immunogenic in vivo . to test this possibility applicant immunized h - 2db −/−, β2m −/−, hla - a2 . 1 + monochain transgenic hhd mice ( 39 ). in these mice the peripheral cd8 + t cell repertoire is essentially educated on the transgenic human molecule . therefore , hhd mice are an excellent tool to assess at the pre - clinical level the ability of individual peptides to induce hla - a2 . 1 restricted ctl responses in vivo ( 25 ). both p540 and p865 were able to induce specific ctl responses ( table ii ) although differences were noted . in fact , p540 induced ctl whether alone or in combination with a helper peptide ( 66 vs . 80 % responders ). in contrast , a high ( 70 %) response against p865 was obtained only when its immunogenicity was increased by association with the helper peptide . the different immunogenicity of the two htrt peptides was also reflected by the magnitude of individual responses ( 55 . 8 ± 9 . 4 vs . 20 ± 11 . 5 % lysis ) against p540 and p865 with helper peptide , respectively . this is consistent with the observation that two normal blood donors responded to immunization against p540 but failed to respond against p865 ( fig1 ). thus , there is an overall correlation between the results of binding / stabilization of the hla - a2 . 1 molecule , the results of immunogenicity in vitro of human pbmc , and the response in vivo in hhd mice . finally , to exclude the development of untoward autoimmunity hhd mice immunized against htrt peptides were monitored with respect to the number of circulating b lymphocytes . using a dual stain ( b220 and anti - ig ) facs analysis applicant found no decrease in circulating b cells in immunized mice when compared to normal hhd mice ( not shown ). furthermore , no enlarged mesenteric lymph nodes nor cellular infiltrates in the liver were noticed after immunization ( not shown ). cancer patients &# 39 ; ctl kill tumor cells of various origins and types because ctl generated against p540 and p865 recognize naturally - processed htrt peptides on lncap prostate cancer cells and htrt activity is expressed at high levels in the table ii induction of ctl against htrt in hla - a2 . 1 transgenic mice helper no . group htrt peptide peptide responders percent lysis i 5401lakflhwl548 − 10 / 15 ( 66 %) ( 35 , 21 , 34 , 42 , 56 , 21 , 12 , 35 , 42 , 16 ) ( seq id no : 1 ) ii 5401lakflhwl548 + 8 / 10 ( 80 %) ( 45 , 56 , 62 , 64 , 65 , 45 , 65 , 45 ) ( seq id no : 1 ) iii 865rlvddfllv873 − 3 / 15 ( 20 %) ( 25 , 12 , 15 ) ( seq id no : 2 ) iv 865rlvddfllv873 + 7 / 10 ( 70 %) ( 25 , 32 , 35 , 12 , 16 , 18 , 2 1 ) ( seq id no : 2 ) a . hhd mice were immunized by a subcutaneous injection of 100 μg of htrt peptide emulsified in incomplete freunds adjuvant ( ifa ). in groups 2 and 4 the htrt peptide was administered together with 140 μg of the helper peptide tppayrppnapil ( seq id no : 4 ) ( 25 ). b . values of cytotoxicity refer to individual responder mice . spleen - derived ctl were harvested 7 days after immunization and then cultured for six days with the homologous htrt peptide . values refer to maximal cytotoxicity at an effector to target ratio of 60 : 1 . vast majority of human cancers , recognition of htrt - derived peptides by ctl could mediate killing of a wide variety of cancer types . ctl lines from a prostate cancer patient were used in a 51 cr - release assay to assess lysis of hla - a2 + tumor cell lines of breast , colon , lung , and melanoma origin as targets . by the trapeze assay all these cell lines were htrt positive . peptide - pulsed t2 cells and lncap prostate cancer cell line served as positive controls ( table iii ). all cell lines but the sw480 colon cell line were lysed by ctl generated against p540 ( range lysis 39 - 48 %). on the other hand , all cell lines but the h69 lung cell line were lysed by ctl generated against p865 ( range lysis 37 - 41 %). the cytotoxic activity was dependent on expression of the hla - a2 molecule since tumor - matched cell lines of a different hla type were not lysed . collectively , these data indicate that htrt peptides such as p540 and p865 are naturally - processed in a variety of tumor cell types . the antigen - recognition activity of t cells is intimately linked with recognition of mhc ( hla in humans ) molecules . this complex is located on chromosome 6 , and encompasses nearly 200 genes encoding for mhc class i and class ii among others . the initial discovery is in relation to the hla - a2 allele , which is expressed in about 50 % of the caucasian population ( 56 ). about 95 % of hla - a2 + white individuals express the hla - a2 . 1 subtype ( 53 ). the majority of peptides bound to mhc class i molecules have a restricted size of 9 ± 1 amino acids and require free n - and c - terminal ends ( 52 ; 59 ; 61 ). in addition to a specific size , different class i molecules appear to require a specific combination of usually two main anchor residues within their peptide ligands ( 52 ; 59 ). in the case of the human allele hla - a2 . 1 , these anchor residues have been described as leucine ( l ) at position 2 and l or valine ( v ) at the c - terminal end ( 52 ). more recently , ruppert et al . found that a “ canonical ” a2 . 1 motif could be defined as l or m ( methionine ) at position 2 and l , v , or i ( isoleucine ) at position 9 ( 60 ). additional criteria were used to refine the selection process . each of the non - anchor table iii percent lysis c htrt ctl ctl cell target cell origin expression a hla - a2 b p540 d 865 d t2 + peptide nd pos . 59 48 t2 nd pos . 11 4 mcf7 breast pos . pos . 39 41 skbr3 pos . neg . 7 9 sw480 colon pos . pos . 12 37 hct011 pos . neg . 9 6 h69 lung pos . pos . 41 9 h146 pos . neg . 11 5 624 melanoma pos . pos . 48 39 1351 pos . neg . 12 6 lncap prostate pos . pos . 44 41 pc3 pos . neg . 9 5 a htrt expression of the tumor cells was determined by a pcr - based assay ( trapezer , intergen ). b expression of hla - a2 was measured by flow cytometry using the monoclonal antibody bb7 . 2 . c cellular cytotoxicity was measured in a 51 cr - release assay at an effector to target ratio of 50 : 1 . all tumor cell lines were incubated with 100 iu / ml of recombinant ifn - γ for 48 hours before the 51 cr - release assay . d patient &# 39 ; s ctl lines 380 . 540 . 1 and 380 . 865 . 1 were generated by immunization with p540 and p865 , respectively . residues ( position 1 , 3 , 4 , 5 , 6 , 7 , 8 ) has significant effect of the a2 . 1 binding ( 60 ). more specifically , some amino acids at position 1 , 3 , 6 , 7 , and 8 virtually abolish a2 . 1 binding capacity of peptides ( 60 ). therefore , applicant excluded all peptides with the following amino acids at the position specified : d ( aspartate ) and p ( proline ) at position 1 ; k ( lysine ) at position 3 ; r ( arginine ) or g ( glycine ) at position 6 ; and e ( glutamate ) at position 7 or 8 . through this selection applicant excluded 12 and retained 27 peptides . by taking into account the frequency of each amino acid in each of the non - anchor positions for many 9mer peptides ( 60 ) applicant defined a more accurate a2 . 1 binders and 10 out of the 27 peptides ( table iv ): the peptide selection was confirmed using the application available online at the web site of the bioinformatics & amp ; molecular analysis section of nih ( bimas . dcrt . nih . gov / molbio / hla - bind / index . html ) that ranks potential 9mer peptides based on a predicted half - time dissociation from hla class i molecules deduced from ( 58 ). in our pilot studies one of the peptides identified using the “ manual ” approach — p865 — ranked among the top hla - a2 - binding peptides identified through the software - guided analysis . another peptide — p540 — ranked at the top in the software - guided analysis . applicant used two such peptides 540ilakflhwl549 ( seq id no : 1 ) and 865rlvddfllv873 ( seq id no : 2 ), denoted as p540 and p865 . both peptides are able to induce a ctl response in vitro in normal blood donors and in patients with prostate cancer . applicant has demonstrated that the same peptides are also able to induce a ctl response in vitro in patients with melanoma . a synopsis of these studies is shown in table v . collectively , it appears that p540 induced a ctl response in 3 out of 4 hla - a2 + patients . p865 induced a response in two patients only . it should be noted that patient 00 was concomitantly being immunized with dendritic cells + melanoma peptides ( peptides other than htrt peptides ) and had a high background making it difficult to decided whether a specific response to htrt had been induced . additional new findings came from exploring the immunogenicity of other htrt peptides . in particular , three peptides were tested whose sequence in the native htrt molecules is shown below in table vi : unlike p540 , which was characterized as having a high affinity binding ( slow half time dissociation ) to hla - a2 ( table vii ), these peptides have an estimated half time dissociation score faster than prototype p540 . calculations we &# 39 ; re made using the program ( bimas . dcrt . nih . gov / molbio / hla_bind / index . html ). applicant then proceeded to add a single residue ( y ) modification in position 1 , which is supposed to increase the binding affinity to hla - a2 and also its immunogenicity ( 60 ). the new modified sequences are shown in table vi . pbmc from three normal hla - a2 + individuals were immunized with the y - modified peptides . the results are summarized as follows ( table vii ). ctl generated against p572 were also able to lyze the htrt +/ hla - a2 + melanoma cell line 624 . the dose response curve of killing of melanoma 624 is shown in fig6 . the antigen - recognition activity of t cells is intimately linked with recognition of mhc ( hla in humans ) molecules . applicant has demonstrated that htrt peptides can expand precursor ctl in pbmc of normal individuals and patients with prostate cancer , and induce in both instances mhc class i - restricted , peptide - specific ctl responses . therefore , the first major implication from these findings is that the available ctl repertoire for htrt is similarly preserved not only in normal individuals as recently reported ( 24 ) but also , and more importantly , in individuals with cancer . this suggests that exposure to cancer does not cause deletion or anergy of clonotypes specific for htrt . since the three patients responding to immunization were resistant to hormone therapy and had metastases , it was surprising that htrt ctl could be induced at such an advanced stage of disease generally characterized by immunosuppression . based on these considerations , one could predict that since the frequency of precursors from pbmc is high enough to permit their expansion in vitro and because htrt peptides bind to mhc class i with sufficient avidity , the two peptides identified in this study may be used for vaccination of hla - a2 + cancer patients . the finding that prostate cancer patients &# 39 ; ctl mediate efficient lysis of a variety of hla - a2 + cancer cells such as prostate , breast , colon , lung and melanoma is unprecedented . based on the values of specific lysis it appears as if these cancer cells are equally effective in processing and presenting the same endogenous htrt peptides . therefore , a second major implication of our study is that similar htrt peptides are expressed and complexed with mhc class i molecules on a variety of cancer cells of different histological origins and types . this renders them susceptible to destruction by ctl and underscores the potential advantage htrt immunization may have in the control of primary tumors and metastases in a large variety of cancer types in humans . the future of htrt - based vaccination will also depend on the type of side effects that may follow immunization . since htrt is expressed in stem cells and mature hemopoietic cells ( 6 , 7 , 38 ), the possibility exists that htrt vaccination could result in autoimmunity and destruction of normal cells . in our hands cancer patients &# 39 ; ctl specific for either p540 or p865 failed to lyse hla - a2 + cd34 + cells . similarly , ctl against p540 raised in normal individuals did not lyse hla - a2 + cd34 + cells ( 24 ). together with the lack of overt autoimmune defects in hemopoietic cells and in the liver in hhd mice following vaccination with htrt peptides , applicant provisionally concludes that ctl specific for htrt are unlikely to trigger autoimmunity against normal cells . possibly , the quantity of htrt peptides generated under physiological lineage / clonotype activation and differentiation is insufficient to mediate lysis by ctl . whether the same holds true for germ cells of reproductive organs for which little is known about cd8 t cell mediated autoimmunity , can only be speculated . while additional experiments are needed , the fact that autoimmunity does not develop after immunization against tumor antigens shared by normal tissues ( 48 , 49 ), including the lymphoid tissue ( 50 ) and reproductive organs ( 51 ), supports the view that htrt - based vaccination in cancer patients may be possible and safe . methods to implement such htrt - based vaccination will include the variety of methods currently in use , such as synthetic peptides , synthetic peptides in immunological adjuvant , dendritic cells pulsed with synthetic peptides , naked dna and rna . in addition , applicant anticipates that effective vaccination can be achieved using transgenic cells . for instance , genes under a specific lymphocyte promoter can be engineered to code for desired htrt peptides , transfected and expressed in lymphocytes from an individual ( e . g ., a cancer patient ), and the patient &# 39 ; s own lymphocytes can be used for vaccination , since lymphocytes process and present peptides to t lymphocytes , hence effecting vaccination . in conclusion , based on the demonstration that precursor ctl specific for two htrt peptides can be expanded in patients with cancer , their ctl recognize the same htrt peptides on tumor cells of various origins and histological types , and a strong in vivo ctl response against both htrt peptides was induced in hla - a2 . 1 + monochain transgenic mice , applicant suggests that htrt can be regarded as a universal cancer , antigen and its peptides as the substrate for a possible universal cancer vaccine for humans . in accordance with the preceding explanation , variations and adaptations of the vaccine and methodology of the present invention will suggest themselves to a skilled practitioner in the medical arts , in the spirit of this invention , these and other possible variations and adaptations of the present invention , and the scope of the invention , should be determined in accordance with the following claims , only , and not solely in accordance with that embodiment within which the invention has been taught . 3 . greider , c . w . 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