Patent Application: US-62317603-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 is 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 ( mh ) antigens , are clearly involved in the development of gvhd after hla - identical bmt . a recent retrospective analysis revealed the significant association between mismatching for the mh antigen ha - 1 and the induction of gvhd after hla - identical bmt 3 . minor histocompatibility antigens 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 mh antigen . the gvhd associated mh antigen 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 mh antigen 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 mh antigen ha - 1 have been isolated from three different patients with severe gvhd 7 . the mh antigen 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 vb6 . 9 and conserved amino acids in the cdr3 region 12 . in a retrospective study , mismatching for a number of mh antigens 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 mh antigen 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 . c .). 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 a 100 different peptides were present in these fractions , around 40 % of fractions 33 and 34 was 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 : ______ ). x stands for isoleucine 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 : ______ ) ( fig2 a ). isoleucine and leucine variants of the peptide were synthesized and run on the microcapillary hplc column . only peptide vlhddllea ( seq id no : ______ ) coeluted with the naturally processed peptide 513 ( results not shown ). next , synthetic vlhddllea ( seq id no : ______ ) added in different concentration to a cml assay with 3 different ha - 1 specific ctl clones revealed recognition by all three clones of the peptide with a half maximal activity at 150 - 200 pm for or all three clones ( fig2 b ). this demonstrated that the mh antigen ha - 1 is represented by the nonapeptide vlhddllea ( seq id no : ______ ). database searches performed to identify the gene encoding ha - 1 , revealed that the ha - 1 peptide vlhdllea ( seq id no : ______ ) was identical for 8 out of 9 amino acids with the peptide vlrddllea ( seq id no : ______ ) 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 : ______ ) 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 amino acid sequence vlhddllea ( seq id no : ______ ) ( 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 mh antigen 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 k1aa0223 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 - 1r 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 mh antigen 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 tnfa 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 mh antigen 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 : ______ ) 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 : ______ ) 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 10 - 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 vlrddllea ( seq id no : ______ ) 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 vlrddllea ( seq id no : 9 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 ha1 - peptide vlrddllea ( seq id no : ______ ) 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 vlrddllea ( seq id no : ______ ) could not be detected ( results not shown ), indicating that this peptide is not or in very low amounts presented by hla - a2 . 1 on the cell surface . this is most likely due to the 10 - 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 mh antigens have been identified so far on the peptide and gene level . two murine mh antigens are encoded by mitochondrial proteins , leading to respectively four and two alleles 15 - 17 , in addition , two murine h - y mh antigens 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 mh antigens 5 , 6 . of the human non - sex linked mh antigens only the mh antigen 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 mh antigen ha - 1 is the first demonstration that human mh antigens 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 mh antigen is the only known human mh antigen 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 mh antigens is probably high , it is envisaged that only few immunodominant mh antigens can account for the risk for gvhd 22 . identification of those human immunodominant mh antigens 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 mh antigen 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 ctl &# 39 ; s . 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 ; minor histocompatibility antigens ( mhags ) 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 fifteen 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 can not be used for generating ctl &# 39 ; s 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 ( neither after ifn - g / tnf - a 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 will spare 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 - 60 days post bmt . it is assumed that most recipient hematopoietic cells are then 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 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 - g and tnf - a treatment of the leukemic cells indicating that cytokines upregulated hla class i expression on the leukemic cells . ha - 1 and ha - 2 specific ctl clones produce ifn - g and tnf - a 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 - a in resistant cases . the feasibility of adoptive immunotherapy with ex - vivo generated ctls depends also 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 9 , 25 and 8 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 2 - 3 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 20u / 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 . [ 0127 ] 51 cr - release assay . hplc fractions and synthetic peptides were tested in a 51 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 minutes 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 h2o , buffer b : 0 . 1 % hfba in acetonitrile . the gradient was 100 % buffer a ( 0 to 20 min ), 0 to 15 % buffer b ( 20 to 25 min ) and 15 to 70 % buffer b ( 25 to 80 min ) at a flow rate of 100 ml / min . 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 h2o , buffer b : 0 . 1 % tfa in acetonitrile . the gradient was 100 % buffer a ( 0 to 20 min ), 0 to 12 % buffer b ( 20 to 25 min ), and 12 to 50 % buffer b ( 25 to 80 min ) at a flow rate of 100 ml / min . 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 min ), 0 to 18 % buffer b ( 29 to 34 min ), 18 % buffer b ( 34 to 39 min ), 18 to 23 . 9 % buffer b ( 39 to 98 min ) at a flow rate of 100 ml / min . 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 . 109 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 min ), 0 to 100 % b ( 5 to 45 min ) at a flow rate of 0 . 5 ml / min . 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 . 1 m acetic acid in water and solvent b was acetonitrile at a flow - rate of 0 . 5 ml / min . one - fifth of the effluent was deposited into the wells of a 96 - well plate containing 100 ml of culture media in each well ( 10 seconds fractions ), while the remaining four - fifths 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 - 27 f to c6 ( flpsdcfpsv ) to recombinant hla - a2 . 1 protein and b2 - microglobulin was used 26 , 27 . in short , hla - a2 . 1 concentrations yielding approximately 40 - 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 1 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 a abimed 422 multiple peptide synthesizer ( abimed , langenfeld , germany ) 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 laboratories , houston , tex .) or according to 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 : ______ ). to amplify the ha - 1 region of kiaa0223 the following primers were used : forward primer 5 ′- gacgtcgtcgaggacatctcccat - 3 ′ ( seq id no : ______ ) and reverse primer 5 ′- gaaggccacagcaatcgtctccagg - 3 ′ ( seq id no : ______ ). cycle parameters used were denaturation 95 ° c ., 1 min , annealing 58 ° c ., 1 min and extension 72 ° c ., 1 min ( 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 ′- ccttga - gaa - act - taa - gga - gtg - tgt - gct - gca - 3 ′ ( seq id no : ______ ), for the ha - 1 r allele primer r1 : 5 ′- cct - tga - gaa - act - taa - gga - gtg - tgt - gtt - gcg - 3 ′ ( seq id no : ______ ) and for both reaction the reverse primer as described above was used . cycle parameters used were denaturation 95 ° c ., 1 min , annealing 67 ° c ., 1 min and extension 72 ° c ., 1 min ( 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 - gcatgg - acg - tcg - tcg - agg - aca - tct - ccc - atc - 3 ′ ( seq id no : ______ ) and a reverse kiaa0223 based pcr primer containing a translational stop signal ( 5 ′- cta - ctt - caggcc - aca - gca - atc - gtc - tcc - agg - 3 ′ ( seq id no : ______ ) 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 ., 1 min , annealing 60 ° c ., 1 min and extension 72 ° c ., 1 min ( 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 3 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 semi automatic multiple peptide synthesizer 4 , 39 . the purity of the peptides was checked by reversed phase high pressure liquid chromatography ( hplc ). 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 h 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 2 - 6 × 10 6 cells with a dc content of 20 - 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 , germany ) by culturing with 100 ng / ml flt3 - ligand ( genzyme , leuven ; belgium ), 30 ng / ml il - 3 , 25 ng / ml scf ( genzyme ) 50 u / ml tnf - a ( genzyme ), 250 u / ml gm - csf ( genzyme ) for 10 to 14 days . the cultures contained 20 - 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 min . at 37 ° c . in serum free aim - v medium . after washing , apc and 10 - 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 an 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 . 10 u / ml of il - 2 was added 24 h . after each restimulation . the t cell lines were expanded with 10 - 20 u / ml il - 2 containing culture medium . cytotoxicity ( 51 cr release ) assays : standard 4 h 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 ) during 72 h . 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 . in short , 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 - g and tnf - a ( both 300 u / ml ) during 72 h . 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 h with or without addition of ifn - g and tnf - a ( 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 twenty - six out of the thirty - five 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 - 1 h 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 - 60 , 000 μm 2 in total ( comprising about 30 - 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 seven 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 seven 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 fifteen 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 twenty - seven 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 ( pn1 - c1 ). 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 ( c . a . klein submitted and 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 - b 14 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 h and ha - 1 r peptides were synthesized using an automated multiple peptide synthesizer ( syro ii , multisyntech , witten , germany ) 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 . 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 : http :// 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 : ______ ) ( 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 . 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 5 min . 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 sec ( 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 o 2 microglobulin ( sigma , st . louis , mo ., usa ). 4 × 10 4 acid treated ebv - lcls were then incubated in 96 - well - v - bottom plates ( costar , cambridge , mass ., usa ) 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 h 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 ., usa ). 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 ). 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 min , 30 min , 45 min as described elsewhere ( 69 - 71 ). the proteolysis products were analyzed by tandem mass spectrometry , as described ( 72 ). 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 ., usa ) and 800 u / ml gmcsf ( donated by dr . s . osanto , lumc , leiden , the netherlands ) for 6 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 2 hours at 37 ° c . in aim - v medium prior to their use as stimulator cells . 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 ., usa ) and 1 u / ml il - 12 ( r & amp ; d systems , minneapolis , minn ., usa ). 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 . 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 - al , - a2 , - a3 , - a11 , - a24 , - b7 , - b8 , - b35 , - b62 . the peptide analysis was extended with two decameric ha - 1h / 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 , - b114 , 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 to hla - b8 or to - b14 ( table 3 ). the decameric ha - 1 h / r peptides vl h / r ddllear 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 , kecvlrddl , kecvlhddll and kecvlrddll 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 3 - 5 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 . 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 besides ha - 1 nonspecific t cells , 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 the 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 - 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 / ha1 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 de 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 - 80 ). in our in vitro cleavage studies , the correct cooh termini of hla - b60 binding sequences of both the ha - 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 4 hr 51cr release assay . the tumor cells from subconfluent cultures were harvested and dispensed at 2500 cells / well in 96 wells flat bottomed microtiter plates and allowed to attach either in the presence or the absence of rinfg ( 250 u / ml , gentech , san francisco , calif .) and tnfa ( 250 u / ml , san francisco , calif .) for 48 hrs . the tumor cells were labeled with 51cr for 1 hr 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 ( bernried , germany ) was used for microdissection 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 10 min . 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 min . to capture mrna . 10 μl cdna wash buffer - 1 ( 50 mm tris - hcl , ph 8 . 3 , 75 mm kcl , 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 : ______ ) and 15 μm cfl5ct ( 5 ′-( ccc ) 5 gtc tag att ( ttt ) 4 tvn ( seq id no : ______ ), at 44 ° c . for 45 min . 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 mgcl2 , 0 . 1 mm dtt , 200 μm gtp ) and cdna - mrna hybrids were denatured at 94 ° c . for 4 min , chilled on ice , 10 u tdt ( mbi - fermentas ) added and incubated at 37 ° c . for 30 - 60 min . after inactivation of the tailing enzyme ( 70 ° c ., 5 min ), 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 : ______ ), 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 . forty cycles were run at 94 ° c . for 15 sec , at 65 ° c ., 30 ° c ., 68 ° c . for 2 min . for the first 20 cycles and a 10 sec - elongation of the extension time each cycle for the remaining 20 cycles , and a final extension step at 68 ° c ., 7 min for expression profiling digoxigenin - utp was incorporated by pcr using 0 . 1 - 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 2 min . followed by 12 cycles at 94 ° c ., 15 sec , 68 ° c ., 3 min . and a final extension time of 7 min . 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 ptg herring sperm to prehybridization buffer , and hybridized for 36 - 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 min 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 ′; reverse : 5 ′- gaa ggc cac agc aat cgt ctc cag - 3 ′ ( seq id no : ______ ) and ha - 1 ( ii ) ( forward : 5 ′- aca ctg ctg tcg tgt gaa gtc - 3 ′ ( seq id no : ______ )); reverse : 5 ′- tca ggc cct gct gta ctg ca - 3 ′ ( seq id no : ______ )). cd45 forward : 5 ′- ctg aag gag acc att ggt ga ( seq id no : ______ )) and reverse 5 ′- ggt act ggt aca cag ttc ga - 3 ′ ( seq id no : ______ ) 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 . [ 0185 ] table 2 ctl recognition of tumor cell lines . the results are given as percentage specific lysis ( example 5 ) by one allo hla - a2 and by two ha - 1 specific ctl clones at different effector ( e ) to target ( t ) ratio &# 39 ; s . % specific lysis by ha - 1 ctls hla - a2 ctls 5w38 3ha15 tumor cell lines ifn γ / tnf α ifn γ / tnf α ifn γ / tnf α tumor type designation e : t no yes no yes no yes breast cancer mda - mb 231 2 : 1 10 13 8 15 8 13 10 : 1 50 64 31 47 25 39 melanoma mel 93 . 04 2 : 1 10 14 1 13 − 2 10 10 : 1 54 64 12 37 17 40 melanoma 453 a0 2 : 1 7 24 1 10 1 18 10 : 1 25 43 5 21 2 22 20 : 1 35 45 7 24 7 21 lung carcinoma glc 36 1 : 1 33 35 6 12 0 8 10 : 1 59 80 8 25 17 25 colon carcinoma caco - 2 1 . 6 : 1 20 22 1 2 6 7 16 : 1 29 49 4 4 11 17 [ 0186 ] table 3 peptides of the ha - 1 polymorphic region tested for binding to different hla class i molecules . ha - 1 s / r polymorphic region sequence binding predicted to peptide # e k l k e c v l h / r d d l l e a r r ( bimas store ) a 1 e k l k e c v l h 2 e k l k e c v l r 3 k l k e c v l h d 4 k l k e c v l r d 5 l k e c v l h d d 6 l k e c v l r d d 7 k e c v l h d d l hla - b60 ( 176 ) 8 k e c v l r d d l hla - b60 ( 176 ) 9 k e c v l h d d l l hla - b60 ( 160 ) 10 k e c v l r d d l l hla - b60 ( 160 ) 11 e c v l h d d l l 12 e c v l r d d l l hla - b8 ( 32 ), - b14 ( 90 ) 13 c v l h d d l l e 14 c v l r d d l l e 15 v l h d d l l e a hla - a2 ( 79 . 6 ) 16 v l r d d l l e a 17 v l h d d l l e a r hla - a3 18 v l r d d l l e a r hla - a3 19 l h d d l l e a r 20 l r d d l l e a r 21 h d d l l e a r r 22 r d d l l e a r r [ 0187 ] table 4 in vitro proteasomal cleavage of a 29 amino acid long ha - 1 a peptide 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 e a % fragment digested in 15 min 30 min 45 min 17 . 2 22 . 4 0 g l e k l k e c v l k d d l 14 . 7 11 . 8 14 . 7 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 13 . 9 16 . 4 21 . 7 h d d l l e a r r p r a h e c l g e a 13 . 0 10 . 3 13 . 9 g l e k l k e c v l h d d l l e a r r p r a 10 . 5 8 . 3 12 . 1 e k l k e c v l h d d l l 9 . 6 8 . 8 12 . 3 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 8 . 5 9 . 2 13 . 8 g l e k l k e c v l h d 7 . 8 7 . 4 11 . 5 g l e k l k e c v l h d d l l e a 4 . 8 5 . 5 0 g l e k l k e c v l [ 0188 ] table 5 in vitro proteasomal cleavage of a 29 amino acidlong ha - 1 r peptide 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 a % fragment digested in 15 min 30 min 45 min 26 . 2 28 . 0 23 . 8 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 14 . 0 l6 . 0 13 . 5 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 11 . 1 14 . 3 12 . 7 g l e k l k e c v l r d d l 7 . 9 9 . 6 7 . 9 g l e k l k e c v l r d d l l e a r r p r a 6 . 6 8 . 6 8 . 3 e k l k e c v l r d d l l 6 . 2 7 . 5 7 . 3 c v l r d d l l e a r r 5 . 3 7 . 0 5 . 7 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 4 . 9 7 . 1 6 . 4 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 4 . 2 6 . 1 5 . 6 g l e k l k e c v l r d 3 . 9 4 . 2 3 . 9 g l e k l k e c v l r d d l l e a 3 . 6 4 . 4 4 . 4 g l e k l k e c v l r d d l l e a r r 3 . 4 4 . 2 3 . 6 g l e k l k e c v l r d d l l e a r r p r 2 . 6 4 . 2 4 . 0 g l e k l k e c v l r d d l l e a r r p r a h [ 0189 ] table 6 ctl analysis nr . of clones dna analysis cell ha - 1 phenotype kiaa0223 sequence sequenced ha - 1 phenotype dh ha - 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