Patent Application: US-70872307-A

Abstract:
peptide sequences constituting t - cell epitopes of minor histocompatibility antigen , ha - 1 . ha - 1 is associated with graft versus host disease . the peptides and their derivatives find many uses , for instance , in bone marrow transplantation , organ transplantation and in treatment of leukemia and non - hematopoietic tumors . the peptide and / or its derivatives can be incorporated in vaccines , in pharmaceutical formulations and they can be used in diagnostic test kits . ha - 1 is expressed by non - hematopoietic tumor cells . while absent in normal epithelial cells , tumor cells and tumor cell lines , particularly from epithelial origin , express ha - 1 and are recognized by ha - 1 cytotoxic t - cells . the invention provides means and methods for ha - 1 specific immunotherapy for ha - 1 - positive patients with non - hematopoietic tumor cells .

Description:
for the sake of illustration , a number of methods and applications are also given below in the examples . gvhd is a frequent and life - threatening complication after allogeneic hla - identical bone marrow transplantation ( bmt ). recipients of hla - identical bone marrow develop acute or chronic gvhd in respectively 36 % and 49 %. 1 , 2 disparities in genes other than the mhc , referred to as minor histocompatibility antigens ( mhags ), are clearly involved in the development of gvhd after hla - identical bmt . a recent retrospective analysis revealed the significant association between mismatching for the mhag ha - 1 and the induction of gvhd after hla - identical bmt . 3 mhags are recognized by mhc restricted t - cells and were shown to be peptides derived from intracellular proteins presented by mhc molecules . 4 - 6 here we report the first identification of a polymorphic gene encoding an human mhag . the gvhd - associated mhag ha - 1 is a nonapeptide derived from the di - allelic kiaa0223 gene . the ha - 1 allelic counterpart encoded by the kiaa0223 gene differs only at one amino acid from the mhag ha - 1 . family studies demonstrated an exact correlation between the kiaa0223 gene polymorphism and the ha - 1 phenotype as was previously determined by recognition by the ha - 1 - specific ctl clones . the elucidation of the ha - 1 encoding gene enables prospective ha - 1 dna typing of bmt donors and recipients to improve donor selection and prevention of gvhd . cytotoxic t - cell clones specific for the mhag ha - 1 have been isolated from three different patients with severe gvhd . 7 the inhag ha - 1 is presented in the context of hla - a2 . 1 and present in 69 % of the hla - a2 . 1 - positive population . 7 ha - 1 expression was demonstrated to be tissue - specific and limited to cells of hematopoietic origin , including dendritic cells , langerhans cells and leukemic cells . 8 - 10 family analysis indicated a mendelian mode of inheritance for ha - 1 and segregation independent from the mhc complex . 11 comparison of the t - cell receptor ( tcr ) sequences of different ha - 1 - specific t - cell clones derived from different individuals revealed conserved usage of the tcr vβ6 . 9 and conserved amino acids in the cdr3 region . 12 in a retrospective study , mismatching for a number of mhags was evaluated with regard to the association with gvhd after hla - identical bmt . a single ha - 1 mismatch between donor and recipient was significantly correlated with the induction of gvhd after hla - identical bmt . 3 to identify the mhag ha - 1 , hla - a2 . 1 molecules were purified from two ha - 1 expressing ebv - transformed b lymphoblastoid cell lines ( ebv - blcl ) rp and blk . the hla - a2 . 1 bound peptides were isolated by acid treatment and fractionation of the peptides was performed by multiple rounds of reverse phase hplc . the fractions were analyzed for their capacity of inducing ha - 1 - specific lysis using t2 cells as target cells and an ha - 1 - specific ctl clone as effector cells in a 51 cr - release assay ( fig1 a ). fraction 24 contained ha - 1 activity and was two times further fractionated with reverse phase hplc using a different organic modifier ( fig1 b and 1c ). fraction 33 and 34 of the third hplc fractionation showed ha - 1 activity 51 cr - release assay and were analyzed by tandem mass spectrometry . because over 100 different peptides were present in these fractions , around 40 % of fractions 33 and 34 were chromatographed with an on - line microcapillary column effluent splitter . the fractions were simultaneously analyzed by tandem mass spectrometry and 51 cr - release assay ( fig1 d ). five peptide species ( at m / z 550 , 520 , 513 , 585 and 502 ) were specifically present in active fractions and absent in fractions without activity in the cml assay . collision - activated dissociation analysis of peptide candidate m / z 550 revealed the sequence yxtdrvmtv ( seq id no : 13 ). x stands for isbleucine or leucine that cannot be discriminated with this type of mass spectrometer . however , a synthetic peptide with this sequence was not able to reconstitute the ha - 1 epitope ( results not shown ). to determine which of the four remaining candidates was the ha - 1 peptide the second ha - 1 purification of the ebv - blcl blk was evaluated . ha - 1 - positive peptide fraction 33 of the second reverse phase hplc fractionation was further chromatographed by microcapillary hplc with a third organic modifier . a single peak of reconstituting activity was observed in a 51 cr - release assay ( results not shown ). mass spectral analysis of these fractions revealed that only peptide candidate m / z 513 was present . this peptide was analyzed with collision - activated dissociation analysis and sequenced as vxhddxxea ( seq id no : 14 ) ( fig2 a ). isoleucine and leucine variants of the peptide were synthesized and run on the microcapillary hplc column . only peptide vlhddllea ( seq id no : 2 ) coeluted with the naturally processed peptide 513 ( results not shown ). next , synthetic vlhddllea ( seq id no : 2 ) added in different concentration to a cml assay with three different ha - 1 - specific ctl clones revealed recognition by all three clones of the peptide with a half maximal activity at 150 to 200 pm for or all three clones ( fig2 b ). this demonstrated that the mhag ha - 1 is represented by the nonapeptide vlhddllea ( seq id no : 2 ). database searches performed to identify the gene encoding ha - 1 , revealed that the ha - 1 peptide vlhdllea ( seq id no : 15 ) was identical for eight out of nine amino acids with the peptide vlrddllea ( seq id no : 10 ) from the kiaa0223 partial complementary dna ( cdna ) sequence , derived from the acute myelogenous leukemia kg - 1 cell line . because ha - 1 has a population frequency of 69 %, we reasoned that vlrddllea ( seq id no : 10 ) might represent the ha - 1 allelic counterpart present in the remaining 31 % of the population . to elaborate on this assumption , we performed cdna sequence analysis of the putative ha - 1 encoding region of kiaa0223 in ebv - blcl derived from a presumed ha - 1 homozygous - positive ( vr ), from a presumed ha - 1 - negative individual ( dh ) and from the kg - 1 cell line ( table 6 .). the ha - 1 encoding region of kiaa0223 of the ha - 1 +/+ individual ( vr ) displayed two nucleotides differences from the kiaa0223 sequence in the databank , leading to the amnino acid sequence vlhddllea ( seq id no : 2 ) ( designated ha - 1 h ). the ha - 1 encoding region of kiaa0223 of the ha - 1 −/− individual ( dh ) showed 100 % homology with the reported kiaa0223 sequence ( designated ha - 1 r ). the kg - 1 cell line expressed both kiaa0223 alleles . because kg - 1 does not express the restriction molecule hla - a2 . 1 necessary for t - cell recognition , we transfected kg - 1 with hla - a2 . 1 and used these cells as target cells in a 51 cr - release assay with the ha - 1 - specific t - cell clone as effector cells . according to the cdna sequence analysis results , the kg - 1 cells were recognized by the ha - 1 - specific t - cell clone ( data not shown ). this result suggested that the kiaa0223 gene forms a di - allelic system of which the ha - 1 h allele leads to recognition by the mhag ha - 1 - specific t - cell clones . two families , who were previously typed for ha - 1 with ha - 1 - specific ctl were studied on the cdna level for their kiaa0223 polymorphism . the family members of family 1 were screened for their kiaa0223 sequence polymorphism by sequencing the ha - 1 encoding sequence region . all ha - 1 - negative members displayed the ha - 1 r sequence , whereas all ha - 1 - positive members turned out to be heterozygous , thus carrying both ha - 1 alleles ( fig3 a ). we subsequently designed ha - 1 allele - specific pcr primers to screen another family previously cellularly typed for ha - 1 . both parents and one child were determined as heterozygous for ha - 1 , two ha - 1 - negative children homozygous for the ha - 1 r allele and one child homozygous for the ha - 1 h allele ( fig3 b ). the screening of both families showed an exact correlation of the ha - 1 phenotype as determined by recognition by the ha - 1 - specific t - cell clones and the kiaa0223 gene polymorphism . to definitely prove that the kiaa0223 gene encodes the mhag ha - 1 , the ha - 1 encoding sequence region of kiaa0223 of both the ha - 1 h and the ha - 1 r alleles were cloned in a eukaryotic expression vector and transiently transfected in ha - 1 - negative hela cells in combination with hla - a2 . 1 . ha - 1 - specific t - cell recognition of these transfected hela cells was assayed using a tnfα release assay . the hela cells transfected with the ha - 1 h sequence containing vector were recognized by two ha - 1 - specific t - cell clones ( fig3 c ). in contrast transfection of the ha - 1 r sequence containing vector did not lead to recognition . in conclusion , our results clearly demonstrate that the mhag ha - 1 is encoded by the ha - 1 h allele of the kiaa023 gene . reconstitution and hla - a2 . 1 binding assays were performed to determine the capacity of ha - 1 r peptide vlrddllea ( seq id no : 10 ) to bind to hla - a2 . 1 and to be recognized by the ha - 1 - specific t - cell clones . the concentration of the ha - 1 r peptide that inhibited the binding of a fluorescent standard peptide to hla - a2 . 1 by 50 % ( ic50 ) was 365 nm , falling in the intermediate binders , whereas the ic50 of the ha - 1 h peptide was 30 nm , which is in the range of high affinity binders ( fig4 a ). 13 , 14 different concentrations of vlrddllea ( seq id no : 10 ) were tested in a 51 cr - release assay with three ha - 1 - specific t - cell clones . one out of the three clones ( 3ha15 ) tested showed recognition of the ha - 1 r peptide , but only at 1000 times higher peptide concentration than that necessary for the recognition of the ha - 1 h peptide ( fig4 b ). as the binding affinity of the two peptides to hla - a2 . 1 differs only ten - fold , it can be concluded that all the t - cell clones specifically recognize the ha - 1 h peptide . the 3ha15 t - cell clone , recognizing the ha - 1 r peptide at high concentrations , does not recognize ha - 1 r homozygous individuals . this suggests that seq id no : 10 is not presented by hla - a2 . 1 or presented below the detection limit of the t - cell . to determine whether the ha - 1 r peptide seq id no : 10 was presented by hla - a2 . 1 , hla - a2 . 1 bound peptides were eluted from an ha - 1 r homozygous ebv - blcl and fractionated with reverse phase hplc . the synthetic ha - 1 peptide seq id no : 10 was run on reverse hplc to determine at which fraction this peptide eluted . the corresponding hplc fractions derived from the ha - 1 r expressing ebv - blcl were analyzed using mass spectrometry . presence of peptide seq id no : 10 could not be detected ( results not shown ), indicating that this peptide is not , or is in very low amounts , presented by hla - a2 . 1 on the cell surface . this is most likely due to the ten - fold lower binding affinity of the peptide for hla - a2 . 1 . the supposed absence of the ha - 1 r peptide in hla - a2 . 1 indicates that this allele must be considered as a null allele with regard to t - cell reactivity . this implicates that only bmt from an ha - 1 r / r ( ha - 1 -) donor to ha - 1 h / h or ha - 1 r / h ( ha - 1 +) recipient direction and not the reverse would be significantly associated with gvhd . this is indeed observed in a retrospective study in which hla - 2 . 1 - positive bmt pairs were typed for ha - 1 . 3 however , ha - 1 r - derived peptides may bind to other hla alleles and possibly be recognized by t - cells . if the latter peptides are not generated and presented by the ha - 1 h allele , then t - cell reactivity towards the ha - 1 r allele may be envisaged and gvhd in that direction may occur . only a few murine and human mhags have been identified so far on the peptide and gene level . two murine mhags are encoded by mitochondrial proteins , leading to respectively four and two alleles . 15 - 17 in addition , two murine h - y mhags were shown to be peptides encoded by y - chromosome located genes . 18 - 21 the human smcy gene , located on the y chromosome , encodes the hla - b7 and the hla - a2 . 1 restricted h - y mhags . 5 , 6 of the human non - sex - linked mhags only the mhag ha - 2 has been sequenced on the peptide level , but the ha - 2 encoding gene remained unknown . 4 the identification of the gene encoding the mhag ha - 1 is the first demonstration that human mhags are derived from polymorphic genes . the ha - 1 encoding kiaa0223 gene has two alleles differing in two nucleotides leading to one single amino acid difference . however , because the kiaa0223 gene has not been fully sequenced yet , it remains to be established whether additional amino acid polymorphisms between the two alleles of this gene are present . because the ha - 1 mhag is the only known human mhag that is correlated with the development of gvhd after bmt the results of our study are of significant clinical relevance . 3 although the numbers of different human mhags is probably high , it is envisaged that only few immunodominant mhags can account for the risk for gvhd . 22 identification of those human immunodominant mhags and screening for those antigens may result in a significant decrease in gvhd after bmt . here , we describe the first elucidation of a polymorphic gene encoding the immunodominant mfag ha - 1 . this enabled us to design ha - 1 allele - specific pcr primers for pre - transplant donor and recipient typing to improve donor selection and thereby prevention of ha - 1 induced gvhd development . it also enabled us to start targeting leukemic cells carrying minor antigens present on hematopoietic cells . one way of arriving at agents targeting leukemic cells , is the ex vivo preparation of ctls . this is explained herein below . allogeneic bone marrow transplantation ( bmt ) is a common treatment of hematological malignancies . 29 recurrence of the underlying malignancy is a major cause of treatment failure . 30 , 31 relapsed cml patients can be successfully treated by donor lymphocyte infusions ( dli ), 32 , 33 but the treatment is less effective for relapsed aml and all , 32 , 33 and is frequently complicated with gvhd . 32 - 34 donor - derived ctls specific for patients &# 39 ; mihags play an important role in both gvhd and gvl reactivities . 10 , 35 - 38 mhags ha - 1 and ha - 2 induce hla - a2 restricted ctls in vivo . mhags ha - 1 and ha - 2 are exclusively expressed on hematopoietic cells including leukemic cells 10 , 36 and leukemic precursors , 37 , 38 but not on cells of the gvhd target organs such as skin fibroblasts , keratinocytes or liver cells . 8 recently the chemical nature of the mhags ha - 1 and ha - 2 was unraveled . 4 , 39 here we report on the feasibility of ex vivo generation of mhag ha - 1 - and ha - 2 - specific ctls from unprimed mhag ha - 1 - and / or ha - 2 - negative healthy blood donors with the purpose of adoptive immunotherapy of relapsed leukemia with a low risk of gvhd . to define the optimal apc for ex vivo generation of ha - 1 - and ha - 2 - specific ctls , we prepared peripheral blood mononuclear cells ( pbmc ), monocytes , peripheral blood circulating dendritic cells ( pbdc ) or dendritic cells derived from bone marrow cd34 + progenitor cells ( bmdc ) from 15 hla - a2 - positive , ha - 1 - or ha - 2 - negative healthy blood donors . these apcs were pulsed with ha - 1 and / or ha - 2 synthetic peptides and used to stimulate autologous unprimed cd8 + t - cells . the attempts to induce ha - 1 - or ha - 2 - specific ctls using monocytes or pbmc were not very successful . pbmc induced in only one out of three attempts ha - 2 - specific ctls . using monocytes , we generated two ha - 1 peptide - specific ctls , but these ctls did not lyse ha - 1 - positive target cells in our experiments ( data not shown ). it is possible that these “ peptide - specific ” ctls have a lower affinity for the naturally expressed ha - 1 antigen , but this does not mean that these cells cannot be used for generating ctls against minor antigens . pbdc were enriched from nine individuals to induce ha - 1 - or ha - 2 - specific ctls . in the four cases where the preparations had a purity of less than 30 % the ctls , lysed peptide loaded target cells , but not mhag - positive target cells ( data not shown ). in contrast , in all cases ( n = 5 ) where pbdc purity was 30 % or more , the ctls not only recognized mhag - negative , peptide pulsed target cells , but also mhag - positive ebv - lcl , demonstrating the recognition of the naturally expressed ligand ( fig1 ). these results underscore the superior capacity of dc to induce t - cell responses from naive precursors and confirm the current opinion . 40 similarly , two bmdc induced ctls that recognized both peptide pulsed target cells and ha - 1 - positive target cells ( fig5 ). no cytotoxic activity was observed against autologous pha stimulated t - cell blasts ( pha blasts ) or against mhag - negative ebv - lcl . thus , neither autoreactivity nor “ third - party ” alloantigen reactivity was observed . several ha - 1 - or ha - 2 - specific ctl clones isolated from these ctls did not react against autologous cells either . these results show that ha - 1 - and ha - 2 - specific ctls can be safely transferred to patients after bmt . the ex vivo induced ha - 1 - and ha - 2 - specific ctls were tested for their hematopoietic cell restricted reactivity and compared with the in vivo induced ha - 1 - and ha - 2 - specific ctls ( fig6 ). pha blasts , but not fibroblasts ( even after ifn - γ / tnf - α stimulation ) were recognized by both ex vivo and in vivo induced ha - 1 - and ha - 2 - specific ctls . fibroblasts , were only lysed after pulsing with the mhag peptides , demonstrating their susceptibility to ctl mediated lysis . these data not only confirm that the ha - 1 and ha - 2 antigens are functionally expressed solely on hematopoietic cells , 8 but also show that adoptive transfer of ha - 1 - or ha - 2 - specific ctls to ha - 1 - or ha - 2 - positive patients spares the patient &# 39 ; s non - hematopoietic tissues and cells . thus , upon adoptive transfer of ha - 1 - and ha - 2 - specific ctls , a low risk of gvhd is to be expected . some precaution may be necessary since we have previously demonstrated that ha - 1 disparity between patient and donor is associated with the development of gvhd in adults . 3 therefore , we transfer the ctls not before 50 to 60 days post bmt . it is assumed that most recipient hematopoietic cells are then to be replaced by donor cells . alternatively , one may transduce the ha - 1 - and ha - 2 - specific ctls with a suicide gene which will make the in vivo elimination of cells possible if adverse effects occur . 41 the ex vivo induced ha - 1 - and ha - 2 - specific ctls were subsequently analyzed for cytolytic activity against , for this study the most relevant target cells , leukemic cells . in vivo induced ha - 1 - and ha - 2 - specific ctls and an hla - a2 - specific alloreactive ctl were used as control effector cells . as shown in fig7 , aml and all cells were lysed by hla - a2 - specific alloreactive ctl , and by in vivo induced ha - 1 - and ha - 2 - specific ctls , indicating that the leukemic cells were positive for hla - a2 and expressed ha - 1 or ha - 2 antigens . as expected the ex vivo induced ctls lysed the leukemic cells comparable to the control effector cells . these results show that ha - 1 - and ha - 2 - specific ctls can also be used as therapy for relapsed aml or all , which are resistant to dli treatment . the level of cytotoxicity could be significantly enhanced following ifn - γ and tnf - α treatment of the leukemic cells indicating that cytokines up - regulated hla class - i expression on the leukemic cells . ha - 1 - and ha - 2 - specific ctl clones produce ifn - γ and tnf - α ex vivo . it is possible that cytokine production by ha - 1 - and ha - 2 - specific ctls occurs in vivo as well . alternatively the efficacy of adoptive immunotherapy with ha - 1 - and ha - 2 - specific ctls may be enhanced by co - administration of ifn - α in resistant cases . the feasibility of adoptive immunotherapy with ex - vivo generated ctls depends on their expandability to sufficient numbers . we , therefore , scored the expansion rates of ha - 1 - and ha - 2 - specific ctls generated by dc . the results indicate that sufficient numbers of ctls for adoptive immunotherapy can be obtained if t - cell cultures will be started with 5 × 10 7 responder cells . for instance , two ha - 2 - specific ctls induced by pbdc showed expansion rates of above nine -, 25 - and eight - fold at the second , third , and fourth week , respectively . these expansion rates translate into an estimated total yield of 3 × 10 9 − 10 10 ctls at the end of the fourth week . the expansion kinetics of the ha - 1 - specific ctls were slower , but the cells expanded consistently with doubling times of two to three days during each restimulation . it is estimated that 10 9 ha - 1 - specific ctls can be obtained after five weeks of culture . in conclusion , our results show for the first time that mhag ha - 1 - and ha - 2 - specific ctls can reproducibly be generated ex - vivo from hla - a2 - positive , mhag ha - 1 - and / or ha - 2 - negative healthy blood donors using dendritic cells pulsed with synthetic peptides . after the successful application of ebv - specific ctls as specific adoptive immunotherapy of ebv - related malignancies , 42 our results now provide a new possibility for the treatment of relapsed , ha - 1 - and / or ha - 2 - positive leukemia patients with ha - 1 - or ha - 2 - specific ctls induced ex - vivo from their hla identical , mhag - negative bone marrow donors . cell culture : the cd8 + hla - a2 . 1 restricted ha - 1 - specific cytotoxic t - cell clones 3ha15 , clone 15 and 5w38 were derived from pbmc of two patients who had undergone hla identical bone marrow transplantation . 7 , 23 the clones were cultured by weekly stimulation with irradiated allogeneic pbmc and blcl in rpmi - 1640 medium containing 15 % human serum , 3 mm 1 - glutamine , 1 % leucoagglutinin - a and 20 u / ml ril - 2 . the hla - a2 . 1 - positive ha - 1 expressing ebv transformed b - cell lines ( blcl ) rp and blk were maintained in imdm containing 5 % fcs . the kg - 1 and t2 cell lines were cultured in 1640 medium containing 3 mm 1 - glutamine and 10 % fcs . 51 cr - release assay : hplc fractions and synthetic peptides were tested in a 5 cr - release assay as described . 24 2500 51 cr labeled t2 cells in 25 ml were incubated with 25 ml peptide dissolved in hanks 50 mm hepes for 30 min . at 37 ° c . cytotoxic t - cells were added in an end volume of 150 ml . when hplc peptide fractions were tested , t2 was incubated with 2 mg / ml ma2 . 1 during the 51 cr labeling . after 4 hours at 37 ° c ., the supernatants were harvested . peptide purification : peptides were eluted out of purified hla - a2 . 1 molecules as earlier described . 24 briefly , hla - a2 . 1 molecules were purified two times from 90 × 10 9 hla - a2 . 1 - positive ebv - blcl by affinity chromatography with bb7 . 2 coupled cnbr - activated sepharose 4b beads ( pharmacia lkb ) and extensively washed . peptides were eluted from the hla - a2 . 1 with treatment with 10 % acetic acid , further acidified by 1 % tfa and separated from the hla - a2 . 1 heavy chain and b2 - microglobulin by filtration over a 10 kd centricon ( amicon ) filter . peptides were fractionated using reverse phase micro hplc ( smart system , pharmacia ). for the first purification , three rounds of hplc fractionation were used to purify the hla - a2 . 1 restricted ha - 1 active peptide fractions from 90 × 10 9 rp cells . the first fractionation consisted of buffer a : 0 . 1 % hfba in h 2 o ; buffer b : 0 . 1 % hfba in acetonitrile . the gradient was 100 % buffer a ( 0 to 20 minutes ), 0 to 15 % buffer b ( 20 to 25 minutes ) and 15 to 70 % buffer b ( 25 to 80 minutes ) at a flow rate of 100 ml / minute . fractions of 100 ml were collected . fraction 24 of the first gradient was further fractionated . the second fractionation consisted of buffer a : 0 . 1 % tfa in h 2 o ; buffer b : 0 . 1 % tfa in acetonitrile . the gradient was 100 % buffer a ( 0 to 20 minutes ), 0 to 12 % buffer b ( 20 to 25 minutes ), and 12 to 50 % buffer b ( 25 to 80 minutes ) at a flow rate of 100 ml / minute . fractions of 100 ml were collected . a shallower third gradient was used to further purify fraction 27 that contained ha - 1 activity . the gradient was 100 % buffer a ( 0 to 29 minutes ), 0 to 18 % buffer b ( 29 to 34 minutes ), 18 % buffer b ( 34 to 39 minutes ), 18 to 23 . 9 % buffer b ( 39 to 98 minutes ) at a flow rate of 100 ml / minute . 1 / 180 to 1 / 45 of the starting material was used to test for positive fractions in the 51 cr - release assay . comparable hplc fractionations were used for the second purification of hla - a2 . 1 restricted ha - 1 active peptide fractions from 90 × 10 9 blk . 40 % of the ha - 1 containing fraction 33 of the second ha - 1 purification was used for an additional reverse phase microcapillary hplc fractionation . buffer a was 0 . 1 % triethyl amine ( tea ) in water buffered to ph 6 . 0 with acetic acid and buffer b was 0 . 085 % tea in 60 % acetonitrile buffered to ph 6 . 0 with acetic acid . the gradient was 100 % buffer a ( 0 to 5 minutes ), 0 to 100 % b ( 5 to 45 minutes ) at a flow rate of 0 . 5 ml / minute . fractions were collected in 50 ml of 0 . 1 % acetic acid every minute for 5 to 15 minutes , every 30 seconds from 15 to 20 minutes , every 20 seconds from 20 to 40 minutes , and every 30 seconds from 40 to 45 minutes . for each fraction collected , 20 % was used to test for ha - 1 activity and 80 % was used to obtain mass spectral data . mass spectrometry : fractions from third dimension hplc separation of the rp purification that contained the ha - 1 activity were analyzed by microcapillary hplc - electrospray ionization mass spectrometry . 25 peptides were loaded onto a c18 microcapillary column ( 75 mm i . d . × 10 cm ) and eluted with a 34 minute gradient of 0 to 60 % b , where solvent a was 0 . 1m hoac ( aq ) and solvent b was acetonitrile at a flow - rate of 0 . 5 ml / minute . 20 % of the effluent was deposited into the wells of a 96 - well plate having 100 ml of culture media in each well ( 10 second fractions ), while the remaining 80 % was directed into the electrospray source of the tsq - 70u . mass spectra and cad mass spectra were recorded on a finnegan - mat tsq - 7000 ( san jose , calif .) triple quadruple mass spectrometer equipped with an electrospray ion source . hla - a2 . 1 peptide binding assay : a quantitative assay for hla - a2 . 1 binding peptides based on the inhibition of binding of the fluorescent labeled standard peptide hbc 18 to 27 ° f . to c6 ( flpsdcfpsv ) ( seq id no : 9 ) to recombinant hla - a2 . 1 protein and b2 - microglobulin was used . 26 , 27 in short , hla - a2 . 1 concentrations yielding approximately 40 to 60 % bound fluorescent standard peptide were used with 15 pmol / well ( 150 nm ) b2 - microglobulin ( sigma ). various doses of the test peptides were coincubated with 100 fmol / well ( 1 nm ) fluorescent standard peptide , hla - a2 . 1 and b2 - microglobulin for one day at room temperature in the dark in a volume of 100 ml in assay buffer . the percent of mhc - bound fluorescence was determined by gel filtration and the 50 % inhibitory dose was deduced for each peptide using one - site competition non - linear regression analysis with the prism graph software . synthetic peptides were manufactured on an abimed 422 multiple peptide synthesizer ( abimed , langenfeld , del .) and were more than 90 % pure as checked by reverse phase hplc . rt - pcr amplification and sequencing of kiaa0223 region coding for ha - 1 . : total or mrna was prepared from blcl using the rnazol method ( cinaa / biotecx labs , houston , tex .) or according to the manufacturer &# 39 ; s instructions ( quickprep mrna purification kit , pharmacia biotech ). cdna was synthesized with 1 mg rna as template and with kiaa0223 based reverse primer 5 ′- gctcctgcatgacgctctgtctgca - 3 ′ ( seq id no : 16 ). to amplify the ha - 1 region of kiaa0223 , the following primers were used : forward primer 5 ′- gacgtcgtcgaggacatctcccat - 3 ′ ( seq id no : 17 ) and reverse primer 5 ′- gaaggccacagcaatcgtctccagg - 3 ′ ( seq id no : 18 ). cycle parameters used were denaturation 95 ° c ., 1 minute , annealing 58 ° c ., 1 minute and extension 72 ° c ., one minute ( 25 cycles ). the pcr - products were purified using the magic pcr - preps dna purification system ( promega ) and direct cloned using the pmosblue t - vector kit ( amersham life science ). six independent colonies from each individual were sequenced using the t7 - sequencing kit ( pharmacia biotech ). ha - 1 allele - specific pcr amplification : in the case of ha - 1 allele - specific pcr amplification , cdna was synthesized as described above . a pcr amplification was performed with allele - specific forward primers : for the ha - 1 h allele primer h1 : 5 ′- cct - tga - gaa - act - taa - gga - gtg - tgt - gct - gca - 3 ′ ( seq id no : 19 ), for the ha - 1 r allele primer r1 : 5 ′- cct - tga - gaa - act - taa - gga - gtg - tgt - gtt - gcg - 3 ′ ( seq id no : 20 ) and for both reactions , the reverse primer as described above was used . cycle parameters used were denaturation 95 ° c ., one minute , annealing 67 ° c ., one minute and extension 72 ° c ., one minute ( 25 cycles ). cloning and expression of ha - 1 h and ha - 1 r allelic region of kiaa0223 : a forward kiaa00223 based pcr primer containing an atg start codon ( 5 ′- ccg - gca - tgg - acg - tcg - tcg - agg - aca - tct - ccc - atc - 3 ′ ( seq id no : 21 )) and a reverse kiaa0223 based pcr primer containing a translational stop signal ( 5 ′- cta - ctt - cag - gcc - aca - gca - atc - gtc - tcc - agg - 3 ′ ( seq id no : 22 )) were designed and used in a rt - pcr reaction with cdna derived from an homozygous ha - 1 h and a homozygous ha - 1 r blcl . cycle parameters used were denaturation 95 ° c ., one minute , annealing 60 ° c ., one minute and extension 72 ° c ., one minute ( 25 cycles ). the desired pcr - products were purified using the magic pcr - preps dna purification system ( promega ). the purified dna was direct cloned using the pmosblue t - vector kit ( amersham life science ) and recloned in the eukaryotic pcdna3 . 1 (+) vector under the control of a cmv promoter . transient cotransfections were performed with hla - a2 . 1 in hela cells using deae - dextran coprecipitation . after three days of culture , ha - 1 - specific t - cells were added and after 24 hours , the tnfa release was measured in the supernatant using wehi cells . 28 peptides : ha - 1 and ha - 2 peptides were synthesized using a semiautomatic multiple peptide synthesizer . 4 , 39 the purity of the peptides was checked by reversed phase high pressure liquid chromatography ( hplc ). apcs : pbmc were isolated by ficoll - hypaque density gradient separation of blood collected with manual hemapheresis . pbdc were enriched from pbmc by depletion of t - cells , monocytes , b - and nk - cells as described earlier . briefly , t - cells were depleted by sheep red blood erythrocyte rosetting . non - t - cells were cultured 36 hours at 37 ° c . in rpmi + 10 % autologous plasma . after depleting monocytes , non - adherent cells were layered on 14 . 5 % metrizamide gradients and centrifuged . the light density pbdc were recovered from the interphase . pbdc were identified by facs being negative for cd3 , cd14 , cd16 and cd19 and positive for hla - dr . the preparations contained two 6 × 10 6 cells with a dc content of 20 to 50 %. in some cases the light density cells were further depleted from cd14 and cd19 cells using antibody coated magnetic beads . bmdc were differentiated from bone marrow cd34 + cells ( isolated using cd34 + isolation kit , macs , bergisch gladbach , de ) by culturing with 100 ng / ml flt3 - ligand ( genzyme , leuven ; be ), 30 ng / ml il - 3 , 25 ng / ml scf ( genzyme ) 50 u / ml tnf - α ( genzyme ), 250 u / ml gm - csf ( genzyme ) for ten to fourteen days . the cultures contained 20 to 60 % dc as detected by high levels of dr and negative expression of cd3 / cd14 / cd16 / cd19 . ex vivo induction of ha - 1 - and ha - 2 - specific ctls : apc were pulsed with ha - 1 or ha - 2 peptides ( both 10 mg / ml ) for 90 minutes at 37 ° c . in serum - free aim - v medium . after washing , apc and ten 15 × 10 6 responder cells ( cd4 depleted autologous pbmc ) were cultured at different apc : responder cell ratios depending on the type of apc ( 5 : 1 , 1 : 3 and 1 : 10 for pbmc , mo and dc , respectively ) in 24 - well culture plates . culture medium was rpmi supplemented with 10 % autologous plasma , 1 u / ml il - 2 ( cetus ), 1 u / ml il - 12 ( genzyme ). the cells were kept at 37 ° c . in a humidified , 5 % co 2 air mixture . at day 5 , 10 u / ml of il - 2 was added . starting from day seven , the t - cell cultures were restimulated weekly with peptide pulsed autologous monocytes . ten u / ml of il - 2 was added 24 hours after each restimulation . the t - cell lines were expanded with 10 to 20 u / ml il - 2 containing culture medium . cytotoxicity ( 51 cr release ) assays : standard 4 - hour 51 cr release assays using pha - blasts , ebv - blcl and fibroblasts and leukemic cells as target cells were performed as described before . 8 the percent specific lysis was calculated using the following formula : 100 ×( cpm experimental release - cpm spontaneous release )/( cpm maximal release - cpm spontaneous release ). target cells : ebv - blcl were generated as described before 8 and cultured in rpmi plus 10 % fcs . pha - activated t - cell blasts ( pha - blasts ) were obtained by stimulation of pbmc with 0 . 1 mg / ml pha ( wellcome ) for 72 hours . pha - blasts were expanded with medium containing 20 u / ml il - 2 . skin fibroblasts of an hla - a2 +, ha - 1 +, ha - 2 + healthy individual were isolated , cultured and tested as described before . 8 fibroblasts were trypsinized and cultured in the wells of 96 - well flat - bottomed microtiter culture plates at a concentration of 3 × 10 3 cells / well with or without addition of ifn - γ and tnf - α ( both 300 u / ml ) for 72 hrs . when indicated , target cells were pulsed with ha - 1 or ha - 2 peptides ( both 10 mg / ml ) during 51 cr labeling . leukemia patients &# 39 ; ( aml or all ) pbmc or bm containing & gt ; 95 % morphologically recognizable malignant cells were assigned as leukemic cells . leukemic cells were thawed and cultured in rpmi plus 10 % human serum for 72 hours with or without addition of ifn - γ and tnf - α ( both 300 u / ml ) before using as target cells . in vivo induced mhag - specific t - cell clones : in vivo induced , mhag ha - 1 - and ha - 2 - specific cd8 + ctl clones were isolated from post bmt leukemia patients , and were documented in detail . 35 to confirm the hematopoietic system restricted tissue distribution , earlier analyzed by ha - 1 - specific ctls , ha - 1 mrna levels were analyzed by quantitative real - time pcr ( example 4 ) in eight different hematopoietic and six different non - hematopoietic cell types . only cells of hematopoietic origin expressed significant levels of the ha - 1 gene ( fig8 ). no significant ha - 1 gene expression was detected in cells of non - hematopoietic origin : i . e ., keratinocytes , dermal fibroblasts , proximal tubular epithelial cells ( ptecs ), human umbilical vein endothelial cells ( huvecs ), melanocytes and sv 40 immortalized breast cell lines hacat and hbl 100 ( fig8 ). next , we investigated the ha - 1 gene transcription levels in 35 epithelial tumor cell lines derived from different carcinomas ( table 1 ). the ha - 1 gene transcription , analyzed by quantitative real time rt - pcr , revealed significant ha - 1 mrna in 26 out of the 35 cell lines of various malignant origins . table 1 also lists the results of the common leukocyte antigen cd45 . we compared the ha - 1 and cd45 rna expression in various hematopoietic cells . both genes are expressed in hematopoietic cells to comparable levels ( data not shown ). none of the tumor cell lines showed significant cd45 gene expression . this shows that ha - 1 transcription observed in the tumor cell lines is specific and not due to contaminating ha - 1 - positive hematopoietic cells ( table 1 ). functional recognition by ha - 1 - specific ctls is a prerequisite for tumor - specific targeting in immunotherapeutic settings . the mhag ha - 1 locus encodes two alleles , i . e ., the ha - 1h and the ha - 1r allele . the ha - 1h allele is the t - cell epitope that is recognized by the hla - a2 restricted ctl . 52 therefore , we executed ctl recognition studies ( example 5 ) on the tumor cell lines that expressed both the hla - a2 restriction molecule and the ha - 1h t - cell epitope required for the hla - a2 restricted ha - 1 - specific ctl recognition . hereto , all tumor cell lines listed in table 1 were hla and ha - 1 genotyped . 52 table 2 shows significant ha - 1 ctl lysis on four of the five cell lines by two ha - 1 - specific clones which could be enhanced in all cases by ifnγ and tnfα treatment of the target cells . the colon carcinoma cell line caco - 2 was only recognized by one of the two ha - 1 ctl clones . with the demonstrated functional expression of ha - 1 by epithelial tumor cell lines , we expected that ha - 1 is also expressed by epithelial tumors in vivo . however , given the expression of ha - 1 by cells of the hematopoietic lineage and in view of the virtual omnipresence of hematopoetic cells in tumors , spurious positive results of a pcr analysis caused by contaminating hematopoietic cells should be avoided . to this end , we applied laser - mediated micro - dissection to cryosections of fresh frozen cancer samples without any microscopically visible leukocyte infiltration ( example 6 , fig9 a ). as control , we used micro - dissected normal breast glands from three patients that underwent breast reduction surgery ( fig9 b ). by the applied micro - dissection method the selected area is cut by a laser beam and directly catapulted into the reaction tube , practically excluding contamination by surrounding tissue . of twelve tumors obtained from patients with breast and lung cancers and the three biopsies from normal breast tissue , areas of 10 , 000 to 60 , 000 μm 2 in total ( comprising about 30 to 200 cells ) were laser - micro - dissected . mrna was isolated , reverse transcribed and amplified with a recently developed global amplification method ( example 7 ). successful global amplification of cdna was checked by established gene - specific amplification of the two housekeeping genes b - actin and ef - 1a and cdna array hybridization ( not shown ). following dilution of the primary pcr products , specific primers served to detect ha - 1 gene expression ( example 8 ). while 7 of twelve tumors were positive for ha - 1 , all normal breast glands were negative ( fig9 c ). the identity of the pcr bands as ha - 1 was confirmed by southern blotting ( not shown ). we used cd45 gene - specific pcr to test whether ha - 1 expression might be attributed to single infiltrating leukocytes or intravascular cells that had escaped our attention . absence of cd45 mrna would provide strong evidence that the ha - 1 signal originates from the epithelial tumor cells in vivo . indeed , four of 7 tumor samples solely expressed ha - 1 ( fig9 c , arrows ) in at least one of the micro - dissected areas , whereas three tumors co - expressed cd45 and ha - 1 prohibiting evaluation of their ha - 1 status . therefore , ha - 1 was found to be expressed in at least 30 % human primary tumors of epithelial origin in vivo . since contamination by cd45 - positive non - epithelial cells could not be absolutely excluded as cause of the encountered cd45 expression in some of the micro - dissected tumor areas , we resorted to ha - 1 analysis of single tumor cells or defined cell clusters freshly isolated from bone marrow or lymph nodes of cancer patients ( fig1 a ). single disseminated cancer cells were detected in cell suspensions prepared from bone marrow and lymph node samples with a fluorescent labeled monoclonal antibody against the epithelial cell adhesion molecule ( epcam ) as marker . 53 in total , twenty - seven single tumor cells or small cell clusters were isolated by micromanipulation from 15 cancer patients ( fig1 a ). for cdna analysis , the same global amplification technique was applied that was used for the micro - dissected tumor areas , enabling faithful detection of expressed transcripts in single cells ( example 7 ). the labeled cdnas were hybridized to an array including specific epithelial marker genes such as the cytokeratin family members ( krt ), mammaglobin ( mbg ) and prolactin induced protein ( pip ) as markers for breast - derived cells , and the transcription factor elf3 . further evidence of epithelial origin was provided by claudin 7 ( cldn7 ) and desmoplakin i ( dsp ) both involved in epithelial cell adhesion . as indicator of malignancy , the expression of mage genes was analyzed , the transcripts are found in spermatogonal cells and exclusively in various cancer cells , hence the designation cancer - testis genes . in addition , we evaluated the cells for markers of hematopoietic cells such as the t - cell receptor , cd45 , cd33 , cd34 , cd37 , cd38 , and cd16 . the isolated cells expressed none of the hematopoietic markers ( not shown ). expression of cytokeratins and other epithelial markers indicated their epithelial origin ( fig1 b ). in some cases the cells were positive for one or more mage genes suggesting their tumor origin , despite down - regulation of cytokeratin mrna ( fig1 b ). all cells were then tested for ha - 1 and cd45 expression by gene - specific pcr ; the ha - 1 amplification products were subsequently confirmed by restriction enzyme digest and by southern blotting ( example 8 ). six of the 27 cells expressed the ha - 1 gene and none of them expressed the cd45 gene ( fig1 ). the ha - 1 significant transcripts were observed in samples derived from breast cancer ( pn4 - c1 ), bronchial carcinoma ( pn3 - c1 , pn5 - c1 , pn6 - c5 ), prostate cancer ( pn2 - c1 ) and cervical cancer ( pn 1 - ci ). from two of the ha - 1 - positive cells ( pn5 - c4 , pn3 - c1 ) we could besides mrna also evaluate their dna by a recently described method . 53 the isolated dna was subjected to whole genome amplification and comparative genomic hybridization ( cgh ). both cells harbored multiple genomic alterations , lending ultimate proof of their malignant nature ( fig1 ). we have investigated whether the ha - 1 h / r polymorphic region contains peptides that can be presented by other hla molecules than hla - a2 . hereto , we analyzed the binding capacities of ha - 1 polymorphic peptides to nine hla - a and - b molecules that have a frequency of more than 10 % in the caucasian population . nonameric ha - 1 h / r peptides ( n = 18 ) were tested for binding to these frequent hla alleles . the peptide binding analyses were extended with two decameric ha - 1 h / r peptides that contained binding motives for hla - a3 and with five nonameric / decameric peptides that were predicted to bind to hla - b14 or to - b60 . next to the binding studies , cellular processing was executed by in vitro proteasome digestion of 29 amino acid long ha - 1 h and ha - 1 r peptides . to enlarge the patient population for ha - 1 - specific immunotherapy , the hla - b60 binding peptides were analyzed for their in vitro immunizing potential . hereto , peptide loaded dendritic cells ( dcs ) were used to induce t - cell responses from healthy individuals . ha - 1 peptides : ha - 1 h and ha - 1 r peptides were synthesized using an automated multiple peptide synthesizer ( syro ii , multisyntech , witten , del .) according to the known ha - 1 amino acid sequence . 61 the purity of the peptides was & gt ; 90 %. the peptides were dissolved in dimethyl sulfoxide ( dmso ), diluted in 0 . 9 % nacl and stored at − 20 ° c . until use . prediction of hla peptide binding : the polymorphic ha - 1 h and ha - 1 r regions were screened with the hla - peptide binding prediction software of bimas ( bioinformatics & amp ; molecular analysis section , nih , bethesda , md . ; url : bimas . dcrt . nih . gov for octameric , nonameric or decameric ha - 1 peptides capable to bind to hla class - i molecules . the selection of peptide candidates was made by comparison of the computed scores with that of the hla - a2 restricted ha - 1 h ctl epitope with amino acid ( aa ) sequence vlhddllea ( seq id no : 2 ) ( score = 79 . 6 ). this score corresponds to the estimated half - time of dissociation of complexes containing the peptide at 37 ° c . at ph 6 . 5 . five ha - 1 h / r peptides with scores ranging from 32 ( intermediate binding score ) to 176 ( strong binding score ) were selected to assay for binding to the relevant hla class - i molecules . the predicted hla class - i / ha - 1 h / r peptide associations and their computed binding scores are presented in table 3 . in addition , we selected two decameric ha - 1 h / r peptides that contained anchor residues for binding to hla - a3 but were not predicted by the bimas software . with reference to table 3 , peptide number 1 is represented by seq id no : 34 , peptide number 2 is represented by seq id no : 35 , peptide number 3 is represented by seq id no : 36 , peptide number 4 is represented by seq id no : 37 , peptide number 5 is represented by seq id no : 38 , peptide number 6 is represented by seq id no : 39 , peptide number 7 is represented by seq id no : 4 , peptide number 8 is represented by seq id no : 5 , peptide number 9 is represented by seq id no : 6 , peptide number 10 is represented by seq id no : 7 , peptide number 11 is represented by seq id no : 40 , peptide number 12 is represented by seq id no : 23 , peptide number 13 is represented by seq id no : 41 , peptide number 14 is represented by seq id no : 42 , peptide number 15 is represented by seq id no : 2 , peptide number 16 is represented by seq id no : 10 , peptide number 17 is represented by seq id no : 43 , peptide number 18 is represented by seq id no : 44 , peptide number 19 is represented by seq id no : 45 , peptide number 20 is represented by seq id no : 46 , peptide number 21 is represented by seq id no : 47 , and peptide number 22 is represented by seq id no : 48 . hla peptide binding assays : we used the competition - based hla peptide binding assay as described previously , with some modifications . 67 briefly , hla typed ebv - lcls were washed with pbs , kept on ice for five minutes and treated with an ice - cold 0 . 132 m citric acid , 0 . 062 m na 2 hpo 4 . 2h 2 o elution buffer for 90 seconds . 67 the ph of the elution buffer was optimized for each hla molecule to enable maximal elution of hla bound peptides ( manuscript in preparation ). immediately after mild acidic treatment , the cells were washed with 12 ml iscove &# 39 ; s modified dulbecco &# 39 ; s medium ( imdm , bio whittaker , belgium ) containing 2 % fcs and resuspended in imdm containing 2 % fcs , 1 . 5 μg / ml β2 microglobulin ( sigma , st . louis , mo ., us ). 4 × 10 4 acid treated ebv - lcls were then incubated in 96 - well v - bottom plates ( costar , cambridge , mass ., us ) with fluorescent - labeled reference peptide ( 25 μl / well , final concentration : 150 nm ) mixed with serial dilutions of competitor ( test ) peptides ( 25 μl / well ; final concentrations : 100 to 0 . 78 μm ) in a total volume of 150 ml . all reference peptides were deduced from previously reported peptides that show strong binding to the respective hla class - i molecules . 68 after incubation for 24 hours at 4 ° c ., the cells were washed twice with 100 μl / well pbs / 1 % fcs and fixed with 0 . 5 % paraformaldehyde in pbs . the mean fluorescence expressed by the cells was determined by a facscalibur flow cytometer ( becton - dickinson , st . louis , mo ., us ). percentage inhibition of the hla binding of the fluorescent reference peptide is calculated with the formula : % inhibition = 1 −[( mean fluorescence in the presence of competitor peptide − mean background fluorescence )/( mean fluorescence in the absence of competitor peptide − mean background fluorescence . )]× 100 %. the relative binding affinity of the peptides is expressed as the peptide concentration that inhibits 50 % of the binding of the reference peptide ( ic 50 ). proteasomal cleavage of the ha - 1 polymorphic region : twenty - nine amino acid long ha - 1 h and ha - 1 r peptides were purified to & gt ; 95 % by reverse phase hplc . 10 μg / ml of the peptides were incubated with 20s proteasomes isolated from ebv - lcls for 15 minutes , 30 minutes ; 45 minutes as described elsewhere . 69 - 71 the proteolysis products were analyzed by tandem mass spectrometry , as described . 72 dendritic cell culture : monocyte - derived dcs ( modcs ) were generated from healthy individuals by culturing peripheral blood - derived cd14 + monocytes with 1000 u / ml il - 4 ( genzyme , cambridge , mass ., us ) and 800 u / ml gmcsf ( donated by dr . s . osanto , lumc , leiden , nl ) for six days as described elsewhere . 73 on day 6 , the dcs were maturated by culturing on irradiated ( 750 gy ) cd40l transfected mouse fibroblasts at a dc to fibroblast ratio of 2 : 1 or by adding 50 % of monocyte conditioned medium . 73 mature dcs were pulsed with ha - 1 peptides for two hours at 37 ° c . in aim - v medium prior to their use as stimulator cells . in vitro induction of hla - b60 / ha - 1 - specific t - cell responses : peptide pulsed dcs were cocultured with autologous pbmc at a dc to pbmc ratio of 1 : 10 in imdm , 10 % human serum supplemented with 1 u / ml il - 2 ( cetus , emeryville , calif ., us ) and 1 u / ml il - 12 ( r & amp ; d systems , minneapolis , minn ., us ). on day 5 , 20 u / ml il - 2 was added . on day 7 , the t - cell lines ( tcl ) were depleted of cd4 + cells using immunomagnetic beads ( dynal as , oslo , norway ) and were restimulated with irradiated ( 150 gy ) peptide pulsed mature dcs ( dc : t - cell ratio 1 : 10 ) or with irradiated ( 150 gy ) peptide pulsed monocytes ( monocyte : t ratio = 1 : 3 ). twenty - four hours and 96 hours after restimulation , medium containing 20 u / ml il - 2 was added . tcl were subsequently restimulated every 7 days and were tested for ha - 1 - specific activity in interferon - γ ( ifn - γ ) elispot assays 74 prior to each restimulation . results : effective binding of nonameric and decameric ha - 1 h and ha - 1 r peptides to hla - b60 : three categories of hla molecules were selected for the peptide binding assays : those molecules with a frequency of more than 10 % in the caucasian population , those with binding motifs and those that were predicted to bind nonameric / decameric ha - 1 h / r peptides . all nonameric ha - 1 h and ha - 1 r peptides ( n = 18 ) were tested for binding to the so called frequent hla class - i molecules hla - a1 , - a2 , - a3 , - a11 , - a24 , - b7 , - b8 , - b35 , - b62 . the peptide analysis was extended with two decameric ha - 1 h / r peptides with a binding motif for hla - a3 and with five nonameric / decameric peptides predicted to bind either to hla - b14 or - b60 ( table 3 ). the hla - a1 , - a11 , - a24 , - b7 , - b8 , - b14 , - b35 and - b62 molecules did not bind nonameric ha - 1 h / r peptides , despite the predictions of bimas software for intermediate to strong binding of peptide ecvlrddll ( seq id no : 23 ) to hla - b8 or to - b14 ( table 3 ). the decameric ha - 1 h / r peptides vlh / rddllear showed weak to intermediate binding to hla - a3 molecules with ic 50 values of 15 . 6 μm and 37 . 5 μm respectively ( fig1 ). in agreement with the prediction of the bimas software , the nonameric and decameric ha - 1 h / r peptides kecvlhddl ( seq id no : 4 ), kecvlrddl ( seq id no : 5 ), kecvlhddll ( seq id no : 6 ) and kecvlrddll ( seq id no : 7 ) showed strong binding to hla - b60 molecules with very low ic 50 values of 5 . 3 μm , 3 . 9 μm , 1 . 0 μm and 1 . 6 μm respectively ( fig1 ). as expected , the original hla - a2 / ha - 1 h ctl epitope , also predicted by the bimas software , displayed binding to hla - a2 with an ic 50 value of 6 . 4 μm ( data not shown ). stable binding of nonameric and decameric ha - 1 h and ha - 1 r peptides to hla - b60 : the stability of the hla - b60 / ha - 1 h / r peptide binding was addressed by testing for the hla peptide binding capacities at 4 ° c . and 25 ° c . hla - a2 / ha - 1 h / r peptide binding stability was analyzed in parallel as comparison . increasing the temperature from 4 ° c . to 25 ° c . did not affect the strong binding of decameric ha - 1 h / r peptides to hla - b60 ( fig1 a ). less binding was observed with the nonameric ha - 1 h / r peptides to hla - b60 ( fig1 b ) which was comparable to the nonameric ha - 1 h peptide to hla - a2 ( fig1 c ). increasing the temperature from 4 ° c . to 25 ° c . further decreased the intermediate binding of the nonameric ha - 1 r peptide to hla - a2 ( fig1 c ). thus , the binding of both ha - 1 h and ha - 1 r peptides to hla - b60 were stable and not temperature sensitive . proper proteasomal cleavage of the hla - b60 binding ha - 1 h / r peptides : twenty - nine amino acid long ha - 1 h / r peptides were subjected to in vitro digestion with ebv - lcl - derived 20s immuno - proteasomes . within a time frame of 15 minutes , major peptide fragments were cleaved at the cooh - termini of both nonameric and decameric hla - b60 binding ha - 1 h / r peptides . the latter cleavage products contained the intact hla - b60 binding sequences with three to five additional amino acid residues at the n termini for the ha - 1 h and ha - 1 r peptides as demonstrated in table 4 and table 5 , respectively . thus , both the ha - 1 h and the ha - 1 r products can be effectively cleaved by proteasomes to generate the precursors of the peptides that bind to hla - b60 . a 29 amino acid long ha - 1 a peptide is represented by seq id no : 78 . fragments 1 through 9 in table 4 are represented by seq id nos : 79 - 87 , respectively . a 29 amino acid long ha - 1 r peptide is represented by seq id no : 88 . fragments 1 through 13 of table 5 are represented by seq id nos : 89 - 101 . in vitro induction of hla - b60 restricted t - cells against the nonameric ha - 1 h peptide : to test the immunogenicity of both the ha - 1 h and the ha - 1 r peptides in the context of hla - b60 , pbmcs from three hla - b60 + ha - 1 rr and from two hla - b60 + ha - 1 hh healthy individuals were stimulated with autologous dcs pulsed with the nonameric ha - 1 h or ha - 1 r peptide , respectively . after two or three rounds of stimulation , the two t - cell lines ( tcl ) induced with the ha - 1 r peptide contained significant numbers of ifn - γ producing t - cells that recognized ha - 1 r peptide pulsed hla - b60 transfected t2 cells . nevertheless , neither tcl induced with ha - 1 r peptide produced ifn - γ upon stimulation with ebv - lcls that express the natural ligand hla - b60 / ha - 1 r ( data not shown ). on the contrary , all three tcl induced with the ha - 1 h peptide contained , aside from ha - 1 non - specific t - cells , a significant number of t - cells that produced ifn - γ not only upon stimulation with ha - 1 h peptide pulsed hla - b60 transfected t2 cells , but also upon stimulation with ebv - lcls that express the natural hla - b60 / ha - 1 h ligand ( fig1 ). in search for novel t - cell epitopes in the ha - 1 h / r polymorphic region , we studied the binding of polymorphic ha - 1 peptides to 11 hla class - i molecules and analyzed the proteasomal cleavage sites in the ha - 1 h / r polypeptides . these analyses suggested novel interactions of both alleles of the mhag ha - 1 locus with hla - b60 molecules . both nonameric and decameric ha - 1 h / r peptides effectively bind to hla - b60 . in vitro proteasomal analysis showed cleavage at the cooh termini of hla - b60 binding peptides , indicating proper intracellular processing . both nonameric and decameric ha - 1 h / r peptides show strong binding to hla - b60 , with ic 50 values between 1 . 6 to 5 . 3 μm . these hla binding levels are similar to or higher than the hla binding of the immunogenic hla - a2 / ha - 1 h ctl epitope and of other reported t - cell epitopes measured in similar assays . 67 , 75 furthermore , we compared the stability of the hla - b60 / ha - 1 h / r with hla - a2 / ha - 1 h / r peptide interactions by increasing the temperature of the binding assays . these assays reveal that unlike the hla - a2 / ha - 1 r peptide interaction , the hla - b60 / ha - 1 h / r and hla - a2 / ha - 1 h interactions are stable . the stability of hla - b60 / ha - 1 h / r interactions were confirmed in separate experiments using fluorescent ha - 1 h / r peptides ( data not shown ). thus , both ha - 1 h and ha - 1 r peptides can efficiently interact with hla - b60 , which is an important biochemical feature of strongly immunogenic t - cell epitopes . 75 this actually predicts immunogenicity of both ha - 1 h and ha - 1 r locus products in association with hla - b60 . the hla peptide binding is preceded by intracellular processing of cellular proteins . in the endoplasmic reticulum ( er ), proteasomally cleaved peptides can undergo nh2 - terminal trimming by aminopeptidases . 76 cooh - terminal trimming in the er have not been demonstrated . the proper generation of the correct cooh terminus by an early major cleavage site by proteasomes is thus a key event for efficient epitope generation as demonstrated by recent studies . 77 - 88 in our in vitro cleavage studies , the correct cooh termini of hla - b60 binding sequences of both the ha - 1 h and the ha - 1 r allele were generated within 15 minutes . these peptide fragments contained the intact hla - b60 binding sequences . the exact sequences of the hla - b60 binding peptides were not present as proteasomal degradation products . also some additional cleavage sites within the putative t - cell epitopes were observed . nonetheless , the successful generation of hla - b60 / ha - 1 h - specific t - cells demonstrates the proper cleavage of the hla - b60 binding ha - 1 h peptides by cellular antigen processing machinery . total rna was prepared from subconfluent layers of the adherent cell cultures using the rnazol method ( cinaa / biotecx laboratories , houston , tex .) according to the manufacturer &# 39 ; s description . cdna was synthesized using 2 mg rna and random hexameric primers . pcr amplification and quantification were performed using the taqman pcr assay ( pe applied biosystems 7700 sequence detector , foster city , calif .). we used comparative quantification normalizing the ha - 1 and cd45 gene to an internal standard gene , the ubiquitously expressed housekeeping gene porphobilinogen deaminase ( pbgd ). to allow calculation of relative levels of expression , we used the kg - 1 cell line , which expresses both genes , as a standard . the ha - 1 and cd45 expression levels of the test samples were calculated as percentages of ha - 1 and cd45 expression levels in the reference cell line kg - 1 . all samples tested which showed expression levels below 10 % in the real time quantitative pcr did not produce detectable pcr fragments in a standard pcr . therefore , expression levels & lt ; 10 % are considered as not significant . the relative quantification was calculated by the linear calibration function between the threshold cycle ( ct ) value and the logarithm of the initial starting quantity ( n ) were ct =− 3 . 31 log ( n )+ 26 . 1 , ct =− 3 . 5 log ( n )+ 21 . 6 and ct =− 3 . 41 log ( n )+ 25 . 6 for ha - 1 , cd45 and pbgd , respectively . the ha - 1 , cd45 and pbgd expression were quantified in all test samples by using these calibration functions . tumor cell lines were used as target cells in a four - hour 51 cr release assay . the tumor cells from subconfluent cultures were harvested and dispensed at 2500 cells / well in 96 - well flat - bottomed microtiter plates and allowed to attach either in the presence or the absence of rinfγ ( 250 u / ml , gentech , san francisco , calif .) and tnfα ( 250 u / ml ) for 48 hours . the tumor cells were labeled with 51 cr for one hour and the experiments were performed in sixplicates . the percentage specific lysis was calculated as follows : % specific lysis =( experimental release / spontaneous release )/( maximal release / spontaneous release )× 100 . preparation of cryosections : sections ( 5 μm ) from freshly shock frozen primary tumors were placed on a polyethylene membrane on a glass slide , stained with meyer &# 39 ; s hematoxylin and dehydrated in 70 %, 90 % and 100 % ethanol . the palm microbeam system ( bemried , del .) was used for micro - dissection and catapulting . detection of disseminated cells , global amplification of micro - dissected areas and of single cells from bone marrow and lymph nodes was performed as described in detail ( klein et al ., submitted ). briefly , the viable bone marrow or lymph node samples were stained for ten minutes with 10 μg / ml monoclonal antibody 3b10 - c9 in the presence of 5 % ab - serum . 3b10 - c9 - positive cells were detected with b - phycoerythrin - conjugated goat antibody to mouse igg ( the jackson laboratory ) and transferred to pcr - tubes on ice . oligo - dt beads in 10 μl lysis buffer ( dynal ) were added , the cells lysed , tubes rotated for 30 minutes to capture mrna . ten μl cdna wash buffer - 1 ( 50 mm tris - hcl , ph 8 . 3 , 75 mm kci , 3 mm mgcl2 , 10 mm dtt , supplemented with 0 . 5 % igepal ( sigma )) was added and mrna bound to the beads washed in 20 μl cdna wash buffer - 2 ( 50 mm tris - hcl , ph 8 . 3 , 75 mm kcl , 3 mm mgcl2 , 10 mm dtt , supplemented with 0 . 5 % tween - 20 ( sigma )), transferred to a fresh tube and washed again in cdna wash buffer - 1 . mrna was reverse transcribed with superscript ii reverse transcriptase ( gibco brl ) using the buffers supplied by the manufacturer supplemented with 500 μm dntp , 0 . 25 % igepal , 30 μm cfl5c8 primer ( 5 ′-( ccc ) 5 gtc tag ann ( n ) 8 - 3 ′ ( seq id no : 25 )) and 15 μm cfl5ct ( 5 ′-( ccc ) 5 gtc tag att ( ttt ) 4 tvn ( seq id no : 26 ), at 44 ° c . for 45 minutes . samples were rotated during the reaction to avoid sedimentation of the beads . cdna remained linked to the paramagnetic beads via the mrna and was washed once in the tailing wash buffer ( 50 mm kh2po4 , ph 7 . 0 , 1 mm dtt , 0 . 25 % igepal ). beads were resuspended in tailing buffer ( 10 mm kh2po4 , ph 7 . 0 , 4 mm mgci2 , 0 . 1 mm dtt , 200 μm gtp ) and cdna - mrna hybrids were denatured at 94 ° c . for four minutes , chilled on ice , 10 u tdt ( mbi - fermentas ) added and incubated at 37 ° c . for 30 to 60 minutes . after inactivation of the tailing enzyme ( 70 ° c ., five minutes ), pcr - mix i was added consisting of 4 μl of buffer - 1 ( roche , taq long template ), 3 % deionized formamide ( sigma ) in a volume of 35 μl . the probes were heated at 78 ° c . in the pcr cycler ( perkin elmer 2400 ), pcr mix ii , containing dntps at a final concentration of 350 μm , cp2 primer ( 5 ′- tca - gaa - ttc - atg - ccc - ccc - ccc - ccc - ccc - 3 ′ ( seq id no : 27 ), final concentration 1 . 2 μm ) and 5 units of the dna poly - mix was added , ( roche , taq long template ) in a volume of 5 μl for a hot start procedure . 40 cycles were run at 94 ° c . for 15 seconds , at 65 ° c ., 30 ° c , 68 ° c . for two minutes for the first 20 cycles and a ten - second elongation of the extension time each cycle for the remaining 20 cycles , and a final extension step at 68 ° c . for 7 minutes . for expression profiling , digoxigenin - utp was incorporated by pcr using 0 . 1 to 1 μl of the original pcr amplified cdna fragments reamplification in the presence of 50 μm dig - dutp ( roche ), 300 μm dttp , and other dntps at a final concentration of 350 μm . reamplification conditions were essentially as described above , modifications were the use of 2 . 5 units of the dna poly mix . initial denaturation at 94 ° c . for two minutes followed by 12 cycles at 94 ° c ., 15 seconds , 68 ° c ., three minutes and a final extension time of 7 minutes . filters were pre - hybridized overnight in the presence of 50 mg / ml e . coli and 50 mg / ml pbs dna in 6 ml dig - easy hyb buffer ( roche ). labeled pcr products from single cells were added in a concentration of 1 . 5 μg / ml mixed with 100 μg herring sperm to prehybridization buffer , and hybridized for 36 to 48 hours . stringency washes were performed according to the roche ™ digoxigenin hybridization protocol adding two final stringency washes in 0 . 1 × ssc + 0 . 1 % sds for 15 minutes at 68 ° c . detection of filter bound probes was performed according to the digoxigenin detection system protocol supplied with the kit ( roche ). amplification of ha - 1 and cd45 . all samples were analyzed by two primer pairs for ha - 1 : ha - 1 ( i ) ( forward : 5 ′- gac gtc gtc gag gac atc tcc cat - 3 ′ ( seq id no : 17 ); reverse : 5 ′- gaa ggc cac agc aat cgt ctc cag - 3 ′ ( seq id no : 18 )) and ha - 1 ( ii ) ( forward : 5 ′- aca ctg ctg tcg tgt gaa gtc - 3 ′ ( seq id no : 29 ); reverse : 5 ′- tca ggc cct gct gta ctg ca - 3 ′ ( seq id no : 30 )). cd45 forward : 5 ′- ctg aag gag acc att ggt ga ( seq id no : 31 )) and reverse : 5 ′- ggt act ggt aca cag ttc ga - 3 ′ ( seq id no : 32 ) primer . amplification products of the ha - 1 ( i ) primers were digested with the restriction enzyme bstu i and amplification products of the ha - 1 ( ii ) primers with hinf i . southern blot was performed according to standard protocols . 1 prediction of hla / peptide associations was executed using bimas software except for peptides 17 and 18 , which were not predicted by bimas but contain hla - 43 anchor amino acids at position 2 and 10 . g l e k l k e c v l h d d l l e a r r p r a h e c l g h d d l l e a r r p r a h e c l g e a g l e k l k e c v l h d d l l e a r r p r a g l e k l k e c v l h d d l l e a r r p r a h e c g l e k l k e c v l h d d l l e a the peptide sequences that bind to hla - b60 are underlined . the proteolytic fragments cleaved at the cooh termini of the hla - b60 binding peptides are indicated in bold . the amounts of the generated fragments after cleavage with 20s immuno proteasomes for 15 , 30 and 45 minutes are expressed as the percentage of all fragments found in the digested substrate . g l e k l k e c v l r d d l l e a r r p r a h e c l g g l e k l k e c v l r d d l l e a r r p r a h e c l g e g l e k l k e c v l r d d l l e a r r p r a g l e k l k e c v l r d d l l e a r r p r a h e c l g l e k l k e c v l r d d l l e a r r p r a h e c g l e k l k e c v l r d d l l e a g l e k l k e c v l r d d l l e a r r g l e k l k e c v l r d d l l e a r r p r g l e k l k e c v l r d d l l e a r r p r a h the peptide sequences that bind to hla - 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